Enhanced immune cell receptor sequencing methods

ABSTRACT

Disclosed are methods for sequencing immune cell receptor repertoires from immune cell populations, the methods comprising isolating RNA from immune cells, generating cDNA from the RNA, ligating adapter sequences to the cDNA, and sequencing the cDNA. Also provided are kits containing primer mixtures for the sequencing of immune cell receptor repertoires.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/481,936, filed Jul. 30, 2019, which is a national stage filing under 35 U.S.C. 371 of International Patent Application Serial No. PCT/US2018/015819, filed Jan. 30, 2018, which claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application Ser. No. 62/452,409, filed Jan. 31, 2017, the entire contents of each of which are incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 5, 2022, is named L046170192US02-SEQ-JRV, and is 150,607 bytes in size.

FIELD

The disclosure relates to methods, systems and kits for sequencing immune cell receptor repertoires from immune cells, such as T-cells or B-cells.

BACKGROUND

Immune cell repertoires, such as B- or T-cell repertoires, consists of millions of lymphocytes, each expressing a different protein complex that enables specific recognition of a single antigen. CD4 and CD8 positive T-cells express so-called T-cell receptors (TCRs). These heterodimeric receptors recognize antigen-derived peptides displayed by major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells, as described in Rudolph M G, Stanfield R L, Wilson I A. How TCRs bind MHCs, peptides, and coreceptors. Annu Rev Immunol. 2006; 24:419-66. TCRs are composed of two subunits, most commonly of one α and one β chain. A less common type of TCR contains one γ and one δ chain.

Alpha (α) chains consists of a (variable) V, a joining (J) and a constant (C) region, while beta (β) chains contain an additional diversity (D) region between the V and the J region (see FIG. 1 ), as described in Starr T K, Jameson S C, Hogquist K A. Positive and negative selection of T cells. Annu Rev Immunol. 2003; 21:139-76. Each of these TCR regions is encoded in several pieces, so-called gene segments, which are spatially segregated in the germline. In humans, the TCR α gene locus contains 54 different V gene segments, and 61 J gene segments. The human TCR β chain locus comprises 65 V, 2 D and 14 J segments. The great structural diversity of TCRs is achieved by somatic recombination of these TCR gene segments during lymphocyte development in the thymus. During this process, several gene segments of each region type are randomly selected and joined to form a rearranged TCR locus. Additional junctional diversity is created by the addition or removal of nucleotides at the sites of recombination, as described in Krangel M S. Mechanics of T cell receptor gene rearrangement. Curr Opin Immunol. 2009 April; 21(2):133-9. The process of V(D)J joining plays a critical role in shaping the third hypervariable loops (also called complementary determining regions, CDR3s) of the TCR α and β chains. These regions bind antigens and are essential for providing the high specificity of antigen recognition that TCRs exhibit.

Similarly to the TCR αβ, TCR gamma (γ) and delta (δ) segments undergo V(D)J rearrangement during thymus development. Both loci are recombined in the double negative (DN) stage of T-cell development. Differentiation towards γδ or αβ lineage relies on the ability of the cell to produce functional γδ or αβ TCR. The δ locus is embedded within the α locus. Dδ, Jδ and Cδ segments are located in between the V and the J segment of the α locus. The Vδ segments are the same as the Vα segments but only a fraction of the Vα segments are used for the TCR δ chain.

Overall, V(D)J recombination is able to generate millions of different TCR sequences and plays a critical role in an organism's ability to eliminate infections or transformed cells. Not surprisingly, TCR repertoires affect a wide range of diseases, including malignancy, autoimmune disorders and infectious diseases. TCR sequencing has been instrumental for our understanding of how the TCR repertoire evolves during infection or following treatment (e.g. after hematopoietic stem cell transplantation, chronical viral infection, immunotherapy). Further, the identification of TCRs on tumor-infiltrating lymphocytes and other T-cells that target cancer-specific epitopes has not only furthered our knowledge of malignant disease, but has also led to novel therapies for cancer such as adoptive T-cell transfer or cancer vaccines.

Due to the large diversity of sequences, determining TCR repertoires has been challenging in praxis. In the last couple of years, next generation sequencing (NGS) has opened up new opportunities to comprehensively assess the extreme diversity of TCR repertoires, as described in Genolet R, Stevenson B J, Farinelli L, Oster{dot over (a)}s M, Luescher I F. Highly diverse TCRα chain repertoire of pre-immune CD8⁺ T cells reveals new insights in gene recombination. EMBO J. 2012 Apr. 4; 31(7):1666-78; Robins H S, Campregher P V, Srivastava S K, Wacher A, Turtle C J, Kahsai O, Riddell S R, Warren E H, Carlson C S. Comprehensive assessment of T-cell receptor beta-chain diversity in alpha beta T cells. Blood. 2009 Nov. 5; 114(19):4099-107; Linnemann C, Heemskerk B, Kvistborg P, Kluin R J, Bolotin D A, Chen X, Bresser K, Nieuwland M, Schotte R, Michels S, Gomez-Eerland R, Jahn L, Hombrink P, Legrand N, Shu C J, Mamedov I Z, Velds A, Blank C U, Haanen J B, Turchaninova M A, Kerkhoven R M, Spits H, Hadrup S R, Heemskerk M H, Blankenstein T, Chudakov D M, Bendle G M, Schumacher T N. High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat Med. 2013 November; 19(11):1534-41; Turchaninova M A, Britanova O V, Bolotin D A, Shugay M, Putintseva E V, Staroverov D B, Sharonov G, Shcherbo D, Zvyagin I V, Mamedov I Z, Linnemann C, Schumacher T N, Chudakov D M. Pairing of T-cell receptor chains via emulsion PCR. Eur J Immunol. 2013 September; 43(9):2507-15.

Since most current TCR sequencing techniques require enrichment of TCR genes for sequencing, the majority of methods include an amplification step, in which the nucleic acids encoding the individual TCRs are amplified. Therefore, one of the challenges of the TCR sequencing relates to the ability of the technology to maintain the proportion of each TCR during the amplification. Thus, the ways in which TCR libraries are prepared have a strong impact on the quality and the reliability of the obtained sequencing results and on the conclusions than can be drawn from the data. Several approaches have been used to amplify and sequence TCR repertoires in the past, each method with its own set of issues.

One frequently employed method for TCR sequencing is based on a multiplex PCR step, in which all the primers for the V and the J segments are mixed together to amplify all the possible V(D)J rearrangements/combinations, as described in Robins H S, Campregher P V, Srivastava S K, Wacher A, Turtle C J, Kahsai O, Riddell S R, Warren E H, Carlson C S. Comprehensive assessment of T-cell receptor beta-chain diversity in alpha beta T cells. Blood. 2009 Nov. 5; 114(19):4099-107. The main drawback of this technology is that the amplification is not quantitative: Because the efficiency of each primer pair varies, some TCR sequences are preferentially represented in the library.

Another TCR sequencing method uses a process called “DNA gene capture” to isolate TCR encoding DNA fragments, as described in Linnemann C, Heemskerk B, Kvistborg P, Kluin R J, Bolotin D A, Chen X, Bresser K, Nieuwland M, Schotte R, Michels S, Gomez-Eerland R, Jahn L, Hombrink P, Legrand N, Shu C J, Mamedov I Z, Velds A, Blank C U, Haanen J B, Turchaninova M A, Kerkhoven R M, Spits H, Hadrup S R, Heemskerk M H, Blankenstein T, Chudakov D M, Bendle G M, Schumacher T N. High-throughput identification of antigen-specific TCRs by TCR gene capture. Nat Med. 2013 November; 19(11):1534-41. However, since this method uses DNA rather than RNA, this method will also isolate V and J segments that have not yet undergone somatic rearrangement. As a consequence, many of the obtained sequencing data are uninformative for TCR gene identification as they do not contain the V(D)J region of rearranged TCR gene locus. Furthermore, using DNA instead of RNA for the TCR gene analysis may overestimate the diversity of the TCR repertoire as only one of the two β chains is expressed by the T-cells while the other gene is silenced (allelic exclusion).

A third method of TCR amplification is based on the 5′-Race PCR technology (SMARTer® Human TCR a/b Profiling Kit, Takara-Clontech). In this method, a nucleic acid adapter is added to the 5′-end of the cDNA during the reverse transcription step. As a result, TCR products can be subsequently amplified with a single primer pair, with one primer binding to the adapter at the 5′-end of the cDNA and the second primer binding to the constant region near the 3′-end of the cDNA. One of the disadvantages of this technique is that the amplification step will generate PCR fragments ranging between 500 and 600 bp. As the length of the V segment exceeds 400 bp it is actually not possible to sequence the V(D)J junction starting from the 5′end using ILLUMINA® sequencing technology, which can generate sequencing reads of up to 300 bp only. Sequencing of the V/J junction is thus usually performed from the constant region, crossing the J segment, the CDR3 region and part of the V segment. However, sequencing errors increase with the length of the sequencing read, and are thus most frequently introduced in the V segments—the region most challenging to correctly assign due to the high homology between different V segments. Consequently, sequencing starting from the constant region may lead to a reduction in the number of V segments that can be identified unambiguously. While this caveat can be avoided by paired-end sequencing, such modification of the protocol will significantly increase the duration and cost associated with this method.

SUMMARY

With each of the current methods exhibiting significant shortcomings, there is thus a considerable need for a TCR sequencing technology that provides TCR repertoire data with high sensitivity and reliability.

Disclosed herein are methods and kits for sequencing of T-cell receptor repertoires and other immune cell repertoires, such as B-cell repertoires, with high sensitivity and reliability. In one embodiment, the methods include the steps of (1) providing RNA from T-cells, (2) transcribing RNA into complimentary RNA (cRNA), (3) reverse transcribing the cRNA into cDNA while introducing a common adapter to the 5′ end of the cDNA products, (4) amplifying the cDNA using a single primer pair, (5) further amplifying with PCR products with a single primer pair which introduces adapters for next generation sequencing, wherein the first primer binds to the common adapter region, and wherein the second primer binds to the constant region of the TCR gene, and (6) sequencing the PCR products. In one embodiment, the methods include the steps of (1) providing RNA from T-cells, (2) reverse transcribing the RNA into cDNA, (3) generating second strand cDNA while introducing a common adapter to the 5′ end of the cDNA products, (4) amplifying the cDNA using a single primer pair, (5) further amplifying with PCR products with a single primer pair which introduces adapters for next generation sequencing, wherein the first primer binds to the common adapter region, and wherein the second primer binds to the constant region of the TCR gene, and (6) sequencing the PCR products. These embodiments are also called SEQTR method (Sequencing T-cell Receptors). Also provided are kits containing primer mixtures for the sequencing of T-cell receptor repertoires. Similar methods and kits for sequencing of B-cell receptor repertoires are provided.

According to one aspect, methods for sequencing immune cell receptor genes are provided. The methods include (1) providing RNA from immune cells; 2)(a) optionally transcribing the RNA into complementary RNA (cRNA), followed by reverse transcribing the cRNA into complementary DNA (cDNA) using one or more primers that comprise a first adapter sequence, wherein each 5′ end of the cDNA produced by reverse transcription contains the first adapter sequence; (2)(b) if step (2)(a) is not performed, reverse transcribing the RNA into complementary DNA (cDNA), followed by transcribing the cDNA into second strand cDNA using one or more primers that comprise a first adapter sequence, wherein each 5′ end of the cDNA produced by transcribing the cDNA into second strand cDNA contains the first adapter sequence; (3) amplifying the cDNA to produce a first amplification product using a first primer pair comprising a first primer that hybridizes to the first adapter sequence and a second primer that hybridizes to a constant region of immune cell receptor gene; (4) amplifying the first amplification product to produce a second amplification product using a second primer pair, in which (i) a first primer of the second primer pair binds to the adapter sequence at the 5′ end of the second amplification product, (ii) the second primer of the second primer pair binds to the constant region of immune cell receptor gene in the second amplification product, and (iii) the first and second primers comprise adapter sequences for sequencing; and (5) sequencing the second amplification product.

In some embodiments, the reverse transcription step results in PCR products ranging from 150-600 bp. In some embodiments, the immune cell receptor genes are T-cell receptor (TCR) genes or B-cell receptor (BCR) genes.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR α chain V segments. In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) comprise one or more of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR β chain V segments. In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) comprise one or more of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR γ chain V segments.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to TCR δ chain V segments.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to BCR heavy chain V segments.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) hybridize to BCR light chain V segments.

In some embodiments, the one or more primers used for reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) contain a nucleotide barcode sequence. In some embodiments, the nucleotide barcode comprises 6 to 20 nucleotides. In some embodiments, the nucleotide barcode consists of 9 nucleotides. In some embodiments, the nucleotide barcode consists of the sequence NNNNTNNNN, NNNNANNNN or HHHHHNNNN.

In some embodiments, the first adapter sequence of the one or more primers used for the reverse transcription (step (2)(a)) or second strand cDNA synthesis (step (2)(b)) comprises a T7 adapter or an ILLUMINA® adapter.

In some embodiments, the immune cells are T-cells and wherein the second primer of the first pair of primers hybridizes to the constant region of a TCR gene.

In some embodiments, the immune cells are B-cells and wherein the second primer of the first pair of primers hybridizes to the constant region of a BCR gene.

In some embodiments, the sequencing is next generation sequencing.

In some embodiments, the RNA from the immune cells is obtained by mixing immune cells with carrier cells before RNA extraction.

In some embodiments, the immune cells are tumor-infiltrating lymphocytes.

In some embodiments, the immune cells are CD4 or CD8 positive T-cells.

In some embodiments, the immune cells are purified from peripheral blood mononuclear cells (PBMC) before RNA extraction.

In some embodiments, the immune cells are part of a mixture of PBMC.

In some embodiments, the immune cells are derived from a mammal. In some embodiments, the mammal is a human or a mouse.

According to another aspect, kits for sequencing of T-cell receptors are provided. The kits include at least one primer which comprises a TCR α chain V segment portion of any one of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310 and a barcode sequence. In some embodiments, the kits include at least one primer including any one of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310.

According to another aspect, kits for sequencing of T-cell receptors are provided. The kits include at least one primer which comprises a TCR β chain V segment portion of any one of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360 and a barcode sequence. In some embodiments, the kits include at least one primer comprising any one of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360.

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the arrangement of (variable) V, diversity (D), joining (J) and constant (C) regions in the α and β chains of T-cell receptors. Figure taken from Murphy, K., Travers, P., Walport, M., & Janeway, C. (2012). Janeway's immunobiology. New York: Garland Science.

FIG. 2 is an illustration of three different TCR sequencing techniques that have been employed in the past.

FIG. 3 provides an overview of the SEQTR method, using TCR α chains as an example. Each bar represents a TCR α chain gene. In RNA and cRNA molecules, the order of the segments is, left to right: V segments, J segments, and the constant region. Barcode regions are added in cDNA molecule to the left of V segments; and T7 adapter regions are added to the left of the barcodes (also indicated by T7 primer amplification in PCR1 and PCR2 steps). ILLUMINA® sequencing adapters are added in the PCR2 step to the 5′ and 3′ ends of the molecules, as shown in the last set of molecules.

FIG. 4 illustrates the sensitivity of the SEQTR method. 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively, were mixed with 5×10{circumflex over ( )}4 3T3 cells. The RNA was extracted and subjected to transcription, reverse transcription and one round of amplification (steps 2-4, see Detailed Description). The resulting PCR products were separated on an agarose gels and visualized with ethidium bromide.

FIG. 5 illustrates the specificity of the SEQTR method. 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively, were mixed with 5×10{circumflex over ( )}4 3T3 cells. The RNA was extracted and subjected to the SEQTR method. The percentages of sequencing reads that were or were not, respectively, associated with actual TCR genes are indicated.

FIG. 6 illustrates the unambiguous identification of TCR genes as a feature of the SEQTR method. 5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively. The RNA of each mixture was isolated and subjected to the SEQTR method. Reads that were not associated with TCR genes were removed from the data set. For the remaining reads, the percentages of reads that could or could not, respectively, be unambiguously assigned to specific V or J segments are indicated.

FIG. 7 illustrates the linearity of the SEQTR method. A fixed amount of DNA encoding a known TCR sequence was diluted at different concentrations into a DNA mixture representing a naïve CD8 T-cell repertoire. TCR repertoires of the individual mixtures were sequenced using the SEQTR method. The observed frequency of the known TCR sequence in the entire repertoire was plotted against the respective TCR gene dilution.

FIG. 8 illustrates the reproducibility of the SEQTR method. TCR repertoires were sequenced using the SEQTR method from one biological sample in two independent technical replicates. The frequencies for each V-J rearrangement/combination in TCR β chains were determined and compared between the two replicates. Each sphere represents a single V-J rearrangement with the size of a sphere indicating the relative frequency of the specific V-J recombination. Grey spheres represent rearrangements for which the relative frequencies detected in the two replicates differed by less than two-fold. Black spheres represent rearrangements for which the relative frequencies detected in the two replicates differed by more than two-fold.

FIGS. 9A-9C illustrates the diversity of three different TCR repertoire sequencing data set using the SEQTR method. FIG. 9A CD8 positive T-cells were isolated from peripheral blood mononuclear cells (PBMC), FIG. 9B additionally purified using tetramers conjugated with neo-epitope TEDYMIHII (SEQ ID NO:236) or FIG. 9C additionally purified using tetramer conjugated with neo-epitope (same as in FIG. 9B) and subsequently expanded in vitro. The TCR repertoires of the respective samples were sequenced using the SEQTR method and the relative frequencies of all observed V/J rearrangements/combinations plotted.

FIGS. 10A-10C illustrate the overlap of TCRs identified using a single cell cloning method and TCRs identified using the SEQTR method. FIG. 10A: T-cells were isolated from PBMC and subjected to an additional round of purification using tetramers conjugated with neo-epitope TEDYMIHII (SEQ ID NO: 236). The resulting cell population was then sorted by fluorescence-activated cell sorting (FACS). Half of the sorted cells were subjected to the SEQTR method to sequence the TCR repertoire. For the other half of cells, individual T-cell clones were isolated and expanded in vitro (single cell cloning). Once the clones were established, the TCR genes of each T-cell clones were amplified and sequenced using classical Sanger sequencing. FIG. 10B: The table shows all six TCRs identified using the single cell cloning method. The sequences correspond to SEQ ID NOs: 237 through 242 from top to bottom, respectively. FIG. 10C: The table shows the eight most frequent TCRs identified using the SEQTR method. The sequences correspond to SEQ ID NOs: 243 through 250 from top to bottom, respectively.

FIG. 11 illustrates the number of reads for different samples obtained using the TCR sequencing service “immunoSEQ®” offered by Adaptive Biotechnology. The requested number of reads per sample was 200,000 reads. The number on the x axis represent analysis of samples from 16 different patients. Columns, left to right, for each sample represent number of reads from: the tumor; the stroma (tissue surrounding the tumor); epitope specific TIL (Tumor Infiltrating Lymphocyte) stained with tetramer and sorted by FACS from the tumor sample (TET); and tetramer sorted TIL from a piece of the tumor that has been engrafted in a mice (mTET).

FIG. 12 illustrates the amplification of TCR genes from T-cells that are part of a PBMC mixture (upper panel) or from isolated, CD4 positive T-cells (lower panel), using steps 2 to 4 of the SEQTR method.

DETAILED DESCRIPTION

In light of the shortcomings of existing techniques to sequence TCRs, it was determined that a TCR sequencing technology providing the most reliable TCR repertoire data includes the following features:

-   -   1) The amplification of TCR genes is linear and does not employ         multiplex PCR, therefore avoiding artificial overrepresentation         of certain TCR sequences.     -   2) The method is based on RNA and not DNA, thus only providing         data for TCR sequences that have undergone rearrangement and         that are actually expressed in T-cells.     -   3) TCR genes are sequenced from the 5′ end, providing high         quality sequencing data and therefore maximizing reliable and         unambiguous identification of the highly homologous V segments.     -   4) Sequencing data include the highly variable CDR3 region,         therefore facilitating unambiguous identification of TCR         sequences.

The disclosed methods, systems and kits fulfill all these criteria. These same features are of use in sequencing receptors from other immune cells, such as B-cells.

In some embodiments, the immune cell receptor sequencing methods comprise the following steps:

-   -   (1) Providing total RNA (RNA) as the starting material;     -   (2)(a) Transcribing the RNA into complimentary RNA (cRNA)         followed by reverse transcribing the cRNA into cDNA, using         primers that introduce a common adapter to the 5′ end of the         cDNA products;     -   (2)(b) If step (2)(a) is not performed, reverse transcribing the         RNA into complementary DNA (cDNA), followed by transcribing the         cDNA into second strand cDNA using one or more primers that         comprise a first adapter sequence, wherein each 5′ end of the         cDNA produced by transcribing the cDNA into second strand cDNA         contains the first adapter sequence;     -   (3) Amplifying the cDNA products using a single primer pair;     -   (4) Amplifying the PCR products of step 4 using a single primer         pair, in which:

i. the primers introduce adapters for next generation sequencing, and ii. the first primer binds to the common adapter region at the 5′ end of the PCR products, and iii. the second primer binds to a region of the PCR products that constitutes the constant region of the TCR to be sequenced; and

-   -   (5) Sequencing the PCR products generated in step 4.

Genetic Information to be Sequenced

The genetic information to be sequenced is immune cell receptor genes. In the some embodiments of the invention, the genetic information to be sequenced comprises T-cell receptors genes. In some embodiments, the TCR genes that are sequenced encode TCR α chains or TCR β chains. In other embodiments, TCR genes that are sequenced encode TCR δ chains or TCR γ chains.

In other embodiments of the invention, the genetic information to be sequenced comprises B-cell receptor (BCR) genes.

Starting Material (Step 1)

RNA is isolated from immune cells and used to generate complimentary RNA (cRNA) by in vitro transcription. This is in contrast to existing TCR sequencing techniques that use DNA or complementary DNA (cDNA) as their genetic starting material.

In some embodiments, the immune cells from which RNA is obtained are isolated from peripheral blood mononuclear cells before RNA extraction. The immune cells are, in some embodiments, T-cells or B-cells.

In some embodiments, T-cells from which RNA is obtained express CD4 or CD8.

Generation of cRNA Through Transcription (Step (2)(a))

Complementary RNA (cRNA) is generated by in vitro transcription. Any method for performing in vitro transcription known to those skilled in molecular biology can be used. In some embodiments, the in vitro transcription in step 2 is performed using commercially available kits, such as the AMBION™ kits available from Thermo Fisher Scientific.

Reverse Transcription (Step (2)(a))

Reverse transcription of the cRNA is performed to generate complementary DNA (cDNA). Methods known to persons skilled in molecular biology are used to reverse transcribe cRNA to cDNA. Typically, such methods include hybridization of a primer to the 3′ end of the cRNA molecule and production of DNA starting at the hybridized primer using a reverse transcriptase enzyme and appropriate nucleotides, salts and buffers.

The choice of primers used in the reverse transcription reaction is important for the ability to differentiate between homologous, yet distinct, immune cell receptor sequences with high degrees of certainty and allows shortening of the V segments from the 5′ end, generating PCR products with a size of 250-300 bp. Such a size range of PCR products is optimal for next generation sequencing.

In some embodiments, the primers used for the reverse transcription are designed to bind within the V segments of the TCR genes (see FIG. 3 ). For example, the reverse transcription primers are designed to bind close enough to the V(D)J junction so that the resulting sequencing data cover the CDR3 of the V segment and the J segment, but far enough from the V(D)J junction to still allow differentiation between different V regions.

In some embodiments of the invention, a set of preferred primers is used (see, e.g., the sequences in Table 2 and Table 4, and Table 8 and Table 9). Due to the high degree of homology between different V segments, some of the primers described in Table 2 and Table 4 (and Table 8 and Table 9) bind to more than one V segment (see Table 3 and Table 5; the binding sites in their respective V segments for primers SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360 are indicated in Table 15 and Table 16). However, the design of the primers presented in Table 2 and Table 4 (likewise Table 8 and Table 9) still allows the unambiguous assignment/identification of the respective V segments based on differences between the V segments downstream of the primer-binding site. In an alternative embodiment of the invention, only a subset of the preferred primers SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360 may be used for the reverse transcription.

In yet another embodiment of the invention, primer sets may be used that bind to different regions in the V segments when compared to the primers having SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360. For instance, the binding site of one or more primers may be moved towards the CDR3 region of the TCR gene. Due to the high degree of homology between V segments, the further the primer binding site is moved in the direction of the CDR3 region of the TCR gene, the larger the likelihood that the resulting sequencing data are consistent with the presence of more than one V segment. While, in these cases, the respective V segments cannot be assigned or identified unambiguously, the number of V/J segments possibly present in the sample can often be narrowed down to a small subset. Depending on the application, such limited information can already be of value to the experimenter.

In another embodiment of the invention, the binding site of one or more primers may be moved towards the 5′ end of the V segment as compared to the binding sites of primers SEQ ID NOs: 1-100 and SEQ ID NOs: 261-360. Many next generation sequencing technologies generate sequencing reads that are 150 bp long. Therefore, the further the primer binding site is moved towards the 5′ end of the V segment, the larger is the probability that the respective J segment (which can be found at the 3′ end of the resulting sequencing read) cannot be identified unambiguously. However, this problem can be circumvented by using alternative sequencing technologies that generate reads >150 bp.

In some embodiments, the primers used in step (2)(a) additionally contain a unique bar code. Such barcoding of each RNA molecule before the amplification can be used to correct the obtained sequencing results for PCR and sequencing errors.

In some embodiments, the primers for this reverse transcription step introduce a common T7 adapter at the 5′ end of the resulting PCR products. However, alternative adapter sequences are possible, including, but not limited to ILLUMINA® adapters and sequences presented in Table 1.

TABLE 1 Examples for alternative nucleotide adapters that can be used instead of a T7 adapter sequence SEQ ID NO Primer name Primer sequence (5′ to 3′) 251 Original Eberwine T7 AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGCT 252 Affymetrix T7 GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGT 253 Invitrogen T7 TAATACGACTCACTATAGGGAGGCGGT 254 Ambion T7 GGTAATACGACTCACTATAGGGAGAAGAGT 255 Agilent T7 AATTAATACGACTCACTATAGGGAGAT Reverse Transcription (Step (2)(b))

Reverse transcription of the RNA is performed to generate complementary DNA (cDNA). Methods known to persons skilled in molecular biology are used to reverse transcribe RNA to cDNA. Typically, such methods include hybridization of a primer to the 3′ end of the RNA molecule and production of DNA starting at the hybridized primer using a reverse transcriptase enzyme and appropriate nucleotides, salts and buffers.

Transcribing the cDNA into Second Strand cDNA (Step (2)(b))

Following generation of cDNA, second strand cDNA is synthesized using methods known to persons skilled in molecular biology. Typically, such methods include hybridization of a primer to the 3′ end of the cDNA molecule and production of second strand cDNA starting at the hybridized primer using a polymerase enzyme and appropriate nucleotides, salts and buffers.

The choice of primers used in the second strand synthesis reaction is step (2)(b) is as described above for reverse transcription in step (2)(a). The choice of primers is important for the ability to differentiate between homologous, yet distinct, immune cell receptor sequences with high degrees of certainty and allows shortening of the V segments from the 5′ end, generating PCR products with a size of 250-300 bp. Such a size range of PCR products is optimal for next generation sequencing.

Amplification (Step 3)

Amplification of the cDNA is performed by any of the well-known amplification reactions, such as polymerase chain reaction (PCR). Methods known to persons skilled in the molecular biology art are used to amplify the cDNA or a portion thereof (e.g., as depicted in FIG. 3 ). Typically, such methods include hybridization of a pair of primers to the cDNA molecule and amplification of the DNA sequence between the hybridized primers using a polymerase enzyme and appropriate nucleotides, salts and buffers.

In some embodiments, the first primer of a primer pair used in an amplification step binds to the common adapter region of the cDNA products produced in step 3 and the second primer of the primer pair binds to a region of the cDNA products that constitutes the constant region of the TCR to be sequenced (see FIG. 3 ).

Of note, not all reverse primers designed to target the constant region of the TCR gene perform equally well in this reaction. For example, the primers listed in Table 7 all failed to provide good amplification with the selected T7 5′ adapter. Therefore, in certain embodiments, the primers listed in are Table 6 used in this amplification step.

Amplification (Step 4)

A second amplification step is performed to add additional sequences to the amplified molecules, such as sequences that are useful in downstream DNA sequencing reactions. In some embodiments of the present invention, the primers used in this step add appropriate adapters for ILLUMINA® sequencing.

Sequencing (Step 5)

Various suitable sequencing methods described herein or known in the art are used to obtain sequence information from the amplified sequences from the nucleic acid molecules within a sample. For example, sequencing methodologies that can be used in the methods disclosed herein include: classic Sanger sequencing, massively parallel sequencing, next generation sequencing, polony sequencing, 454 pyrosequencing, ILLUMINA® sequencing, SOLEXA® sequencing, SOLID™ sequencing (sequencing by oligonucleotide ligation and detection), ion semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real time sequencing, nanopore DNA sequencing, tunneling currents DNA sequencing, sequencing by hybridization, sequencing with mass spectrometry, microfluidic Sanger sequencing, microscopy-based sequencing, RNA polymerase sequencing, in vitro virus high-throughput sequencing, Maxam-Gilbert sequencing, single-end sequencing, paired-end sequencing, deep sequencing, and/or ultra-deep sequencing.

Definitions

As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

As used herein, a “primer” is a nucleic acid molecule that hybridizes with a complementary (including partially complementary) polynucleotide strand. Primers can be DNA molecules, RNA molecules, or DNA or RNA analogs. DNA or RNA analogs can be synthesized from nucleotide analogs.

EXAMPLES Example 1: Exemplary Protocol for the SEQTR Method Using T7 and TrueSeq Adapters

TCR α and β chain genes were sequenced in two independent reactions.

1) Starting material and RNA extraction

-   -   To obtain sufficient amounts of RNA in the extraction, a minimum         of 500,000 T-cells were used as starting material.         Alternatively, and especially in instances where fewer T-cells         were available, T-cells were mixed with 50,000 mouse 3T3 cells         that served as carrier. T-cell RNA was extracted using the         RNeasy® Micro Kit from Qiagen Inc. according the manufacturer's         instruction with the following modification: Elution was         performed with 20 μl of water preheated to 50° C. RNA quality         and quantity was verified using a fragment analyzer.

2) cRNA synthesis by in vitro transcription (IVT):

-   -   In vitro transcription of isolated RNA was performed using the         MessageAmp™ II aRNA Amplification Kit from Ambion® (Thermo         Fisher Scientific), which contains enzymes, buffers and         nucleotides required to perform the first and second strand cDNA         and the in vitro transcription. The kit also provides all         columns and reagents needed for the cDNA and cRNA purifications.         RNA amplification was performed according to the manufacturer's         instructions with the following modifications: 1) Between 0.5         and 1 μg of total RNA as was used as starting material. 2) The         IVT was performed in a final volume of 40 μl, and incubated at         37° C. for 16 h. Purified cRNA was quantified by absorbance         using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific).

3) cDNA synthesis by reverse transcription:

-   -   The reverse transcription of the cRNA was performed with the         SuperScript® III from Invitrogen (Thermo Fisher Scientific). The         kit provides the enzyme, the buffer and the dithiothreitol (DTT)         needed for the reaction. Deoxynucleotides (dNTPs) and RNAsin®         Ribonuclease inhibitor were purchased from Promega. The         sequences for the primers used for the reverse transcription can         be found in Table 2 (primers for sequencing TCR α chain genes)         and Table 4 (primers for sequencing TCR β chain genes).     -   500 ng of cRNA were used as starting material for the reverse         transcription. cRNA was mixed with 1 μl hTRAV or hTRBV primers         mix (2 μM each) and 1 μl dNTP (25 mM) in a final volume of 13         μl. The mix was first incubated at 70° C. for 10 min, then at         50° C. for 30 s. 4 μl 5× buffer, 1 μl DTT (100 mM), 1 μg         SuperScript III and 1 μl RNAsin® were added to the mix. The         samples were subsequently incubated for at 55° C. 1 h and then         at 85° C. for 5 min. After the cDNA synthesis, 1 μg DNase-free         RNase (Roche) was added to the cDNA and incubated at 37° C. for         30 min to remove the cRNA.

4) TCR gene amplification:

-   -   TCR gene amplification was performed using a Phusion®         High-Fidelity DNA polymerase (New England Biolabs) under the         following conditions: PCR mix: 1 μl cDNA from step 3, 1 μl dNTPs         (25 mM), 1 μl primer mix (10 μM each, see Table 5), 5 μl 5×         buffer and 0.2 μl Phusion® enzyme in a total volume of 25 μl.     -   PCR conditions:         -   94° C. for 5 min         -   20 to 30 cycles of             -   98° C. for 10 s             -   55° C. for 30 s             -   72° C. for 30 s         -   72° C. for 2 min     -   PCR products were purified either from agarose gels (using a         Qiaquick Gel Extraction Kit from Qiagen) or using an ExoSAP-IT®         PCR Product Cleanup Kit (Affymetrix) according to the         manufacturer's instructions.

5) Addition of Next Generation Sequencing adapters:

-   -   ILLUMINA® sequencing adapters were added by PCR using a Phusion®         High-Fidelity DNA polymerase (New England Biolabs). One third of         the purified PCR product obtained in step 4 was mixed with 0.5         μl dNTPs (25 mM), 1 μl primer mix (10 μM each, see Table 8), 5         μl 5× buffer and 0.2 μl Phusion® enzyme in a total volume of 25         μl.     -   PCR conditions:         -   94° C. for 5 min         -   perform 12 cycles of:             -   98° C. for 10 s             -   55° C. for 30 s             -   72° C. for 30 s         -   72° C. for 2 min

6) TCR library purification:

-   -   10 μl of the PCR product from step 5 were purified using an         ExoSAP-IT® PCR Product Cleanup Kit (Affymetrix) or Ampure XP         beads (Beckman Coulter) according to the manufacturer's         instruction. Samples could then directly be used for ILLUMINA®         sequencing.

TABLE 2 Preferred primer sequences for amplification of TCR a chain V segments. N can be any  nucleotide. The sequences for primers presented in this table consist of three parts (listed from 5′ to 3′): T7 adapter, barcode and TCR a chain V segment. Sequence SEQ Sequence T7 adapter barcode portion Sequence TCR a chain V ID NO Primer name portion of the primer of the primer segment portion of the primer  1 hTRAV1-1 TGTAATACGACTCACTATAG NNNNTNNNN CTTCTACAGGAGCTCCAGATGAAAG  2 hTRAV1-2 TGTAATACGACTCACTATAG NNNNTNNNN CTTTTGAAGGAGCTCCAGATGAAAG  3 hTRAV2 TGTAATACGACTCACTATAG NNNNTNNNN TGCTCATCCTCCAGGTGCGGGA  4 hTRAV3 TGTAATACGACTCACTATAG NNNNTNNNN GAAGAAACCATCTGCCCTTGTGA  5 hTRAV4 TGTAATACGACTCACTATAG NNNNTNNNN CCTGCCCCGGGTTTCCCTGAGCGAC  6 hTRAV5 TGTAATACGACTCACTATAG NNNNTNNNN TCTCTGCGCATTGCAGACACCCA  7 hTRAV6 TGTAATACGACTCACTATAG NNNNTNNNN TTGTTTCATATCACAGCCTCCCA  8 hTRAV7 TGTAATACGACTCACTATAG NNNNTNNNN GCTTGTACATTACAGCCGTGCA  9 hTRAV8-1/8-3 TGTAATACGACTCACTATAG NNNNTNNNN ATCTGAGGAAACCCTCTGTGCA 10 hTRAV8-2/8-4 TGTAATACGACTCACTATAG NNNNTNNNN ACCTGACGAAACCCTCAGCCCAT 11 hTRAV8-5 TGTAATACGACTCACTATAG NNNNTNNNN CCTATGCCTGTCTTTACTTTAATC 12 hTRAV8-6 TGTAATACGACTCACTATAG NNNNTNNNN CTTGAGGAAACCCTCAGTCCATAT 13 hTRAV8-7 TGTAATACGACTCACTATAG NNNNTNNNN GAAACCATCAACCCATGTGAGTGA 14 hTRAV9-1 TGTAATACGACTCACTATAG NNNNTNNNN ACTTGGAGAAAGACTCAGTTCAA 15 hTRAV9-2 TGTAATACGACTCACTATAG NNNNTNNNN ACTTGGAGAAAGGCTCAGTTCAA 16 hTRAV10 TGTAATACGACTCACTATAG NNNNTNNNN CTGCACATCACAGCCTCCCA 17 hTRAV11 TGTAATACGACTCACTATAG NNNNTNNNN GTTTGGAATATCGCAGCCTCTCAT 18 hTRAV12-1 TGTAATACGACTCACTATAG NNNNTNNNN CCCTGCTCATCAGAGACTCCAAG 19 hTRAV12-2 TGTAATACGACTCACTATAG NNNNTNNNN CTCTGCTCATCAGAGACTCCCAG 20 hTRAV12-3 TGTAATACGACTCACTATAG NNNNTNNNN CCTTGTTCATCAGAGACTCACAG 21 hTRAV13-1 TGTAATACGACTCACTATAG NNNNTNNNN TCCCTGCACATCACAGAGACCCAA 22 hTRAV13-2 TGTAATACGACTCACTATAG NNNNTNNNN TCTCTGCAAATTGCAGCTACTCAA 23 hTRAV14 TGTAATACGACTCACTATAG NNNNTNNNN TTGTCATCTCCGCTTCACAACTGG 24 hTRAV15 TGTAATACGACTCACTATAG NNNNTNNNN GTTTTGAATATGCTGGTCTCTCAT 25 hTRAV16 TGTAATACGACTCACTATAG NNNNTNNNN CCTGAAGAAACCATTTGCTCAAGA 26 hTRAV17 TGTAATACGACTCACTATAG NNNNTNNNN TCCTTGTTGATCACGGCTTCCCGG 27 hTRAV18 TGTAATACGACTCACTATAG NNNNTNNNN ACCTGGAGAAGCCCTCGGTGCA 28 hTRAV19 TGTAATACGACTCACTATAG NNNNTNNNN CACCATCACAGCCTCACAAGTCGT 29 hTRAV20 TGTAATACGACTCACTATAG NNNNTNNNN TTTCTGCACATCACAGCCCCTA 30 hTRAV21 TGTAATACGACTCACTATAG NNNNTNNNN CTTTATACATTGCAGCTTCTCAGCC 31 hTRAV22 TGTAATACGACTCACTATAG NNNNTNNNN GTACATTTCCTCTTCCCAGACCAC 32 hTRAV23 TGTAATACGACTCACTATAG NNNNTNNNN CATTGCATATCATGGATTCCCAGC 33 hTRAV24 TGTAATACGACTCACTATAG NNNNTNNNN GCTATTTGTACATCAAAGGATCCC 34 hTRAV25 TGTAATACGACTCACTATAG NNNNTNNNN CAGCTCCCTGCACATCACAGCCA 35 hTRAV26-1 TGTAATACGACTCACTATAG NNNNTNNNN TTGATCCTGCCCCACGCTACGCTGA 36 hTRAV26-2 TGTAATACGACTCACTATAG NNNNTNNNN TTGATCCTGCACCGTGCTACCTTGA 37 hTRAV27 TGTAATACGACTCACTATAG NNNNTNNNN GTTCTCTCCACATCACTGCAGCC 38 hTRAV28 TGTAATACGACTCACTATAG NNNNTNNNN GCCACCTATACATCAGATTCCCA 39 hTRAV29 TGTAATACGACTCACTATAG NNNNTNNNN TCTCTGCACATTGTGCCCTCCCA 40 hTRAV30 TGTAATACGACTCACTATAG NNNNTNNNN CCCTGTACCTTACGGCCTCCCAGCT 41 hTRAV31 TGTAATACGACTCACTATAG NNNNTNNNN CTTATCATATCATCATCACAGCCA 42 hTRAV32 TGTAATACGACTCACTATAG NNNNTNNNN TCCCTGCATATTACAGCCACCCAA 43 hTRAV33 TGTAATACGACTCACTATAG NNNNTNNNN ACCTCACCATCAATTCCTTAAAAC 44 hTRAV34 TGTAATACGACTCACTATAG NNNNTNNNN TCCCTGCATATCACAGCCTCCCAG 45 hTRAV35 TGTAATACGACTCACTATAG NNNNTNNNN CTTCCTGAATATCTCAGCATCCAT 46 hTRAV36 TGTAATACGACTCACTATAG NNNNTNNNN TCCTGAACATCACAGCCACCCAG 47 hTRAV37 TGTAATACGACTCACTATAG NNNNTNNNN TCCCTGCACATACAGGATTCCCAG 48 hTRAV38 TGTAATACGACTCACTATAG NNNNTNNNN CAAGATCTCAGACTCACAGCTGG 49 hTRAV39 TGTAATACGACTCACTATAG NNNNTNNNN CCGTCTCAGCACCCTCCACATCA 50 hTRAV40 TGTAATACGACTCACTATAG NNNNTNNNN CCATTGTGAAATATTCAGTCCAGG

TABLE 3 V segments targeted by each primer used for the amplification of TCR α chain V segments. SEQ ID NO Primer Targeted V segment(s) 1 hTRAV1-1 hTRAV01-1 2 hTRAV1-2 hTRAV01-2 3 hTRAV2 hTRAV02 4 hTRAV3 hTRAV03 5 hTRAV4 hTRAV04 6 hTRAV5 hTRAV05 7 hTRAV6 hTRAV06 8 hTRAV7 hTRAV07 9 hTRAV8-1/8-3 hTRAV08-1, hTRAV08-3 10 hTRAV8-2/8-4 hTRAV08-2, hTRAV08-4 11 hTRAV8-5 hTRAV08-5 12 hTRAV8-6 hTRAV08-6 13 hTRAV8-7 hTRAV08-7 14 hTRAV9-1 hTRAV09-1 15 hTRAV9-2 hTRAV09-2 16 hTRAV10 hTRAV10, hTRAV41 17 hTRAV11 hTRAV11 18 hTRAV12-1 hTRAV12-1 19 hTRAV12-2 hTRAV12-2 20 hTRAV12-3 hTRAV12-3 21 hTRAV13-1 hTRAV13-1 22 hTRAV13-2 hTRAV13-2 23 hTRAV14 hTRAV14 24 hTRAV15 hTRAV15 25 hTRAV16 hTRAV16 26 hTRAV17 hTRAV17 27 hTRAV18 hTRAV18 28 hTRAV19 hTRAV19 29 hTRAV20 hTRAV20 30 hTRAV21 hTRAV21 31 hTRAV22 hTRAV22 32 hTRAV23 hTRAV23 33 hTRAV24 hTRAV24 34 hTRAV25 hTRAV25 35 hTRAV26-1 hTRAV26-1 36 hTRAV26-2 hTRAV26-2 37 hTRAV27 hTRAV27 38 hTRAV28 hTRAV28 39 hTRAV29 hTRAV29 40 hTRAV30 hTRAV30 41 hTRAV31 hTRAV31 42 hTRAV32 hTRAV32 43 hTRAV33 hTRAV33 44 hTRAV34 hTRAV34 45 hTRAV35 hTRAV35 46 hTRAV36 hTRAV36 47 hTRAV37 hTRAV37 48 hTRAV38 hTRAV38-1, hTRAV38-2 49 hTRAV39 hTRAV39 50 hTRAV40 hTRAV40

TABLE 4 Preferred primer sequences for amplification of TCR 3 chain V segments. N can be any nucleotide. The sequences for primers presented in this table consist of three parts (listed from 5′ to 3′): T7 adapter, barcode and TCR 3 chain V segment. Sequence SEQ Sequence T7 adapter barcode portion Sequence TCR 3 chain V segment ID NO Primer name portion of the primer of the primer portion of the primer  51 hTRBV1 TGTAATACGACTCACTATAG NNNNANNNN GTGGTCGCACTGCAGCAAGAAGA  52 hTRBV2 TGTAATACGACTCACTATAG NNNNANNNN GATCCGGTCCACAAAGCTGGAGGA  53 hTRBV3-1 TGTAATACGACTCACTATAG NNNNANNNN CATCAATTCCCTGGAGCTTGGTGA  54 hTRBV4-1 TGTAATACGACTCACTATAG NNNNANNNN TTCACCTACACGCCCTGCAGCCAG  55 hTRBV4-2 TGTAATACGACTCACTATAG NNNNANNNN TTCACCTACACACCCTGCAGCCAG  56 hTRBV5-1 TGTAATACGACTCACTATAG NNNNANNNN GAATGTGAGCACCTTGGAGCTGG  57 hTRBV5-2 TGTAATACGACTCACTATAG NNNNANNNN TACTGAGTCAAACACGGAGCTAGG  58 hTRBV5-3 TGTAATACGACTCACTATAG NNNNANNNN GCTCTGAGATGAATGTGAGTGCCT  59 hTRBV5-4 TGTAATACGACTCACTATAG NNNNANNNN CTGAGCTGAATGTGAACGCCTT  60 hTRBV6-1 TGTAATACGACTCACTATAG NNNNANNNN GAGTTCTCGCTCAGGCTGGAGT  61 hTRBV6-2 TGTAATACGACTCACTATAG NNNNANNNN CTGGGGTTGGAGTCGGCTGCTC  62 hTRBV6-4 TGTAATACGACTCACTATAG NNNNANNNN CCCCTCACGTTGGCGTCTGCTG  63 hTRBV6-5 TGTAATACGACTCACTATAG NNNNANNNN TCCCGCTCAGGCTGCTGTCGGC  64 hTRBV6-6 TGTAATACGACTCACTATAG NNNNANNNN GATTTCCCGCTCAGGCTGGAGT  65 hTRBV6-7 TGTAATACGACTCACTATAG NNNNANNNN TCCCCCTCAAGCTGGAGTCAGCT  66 hTRBV6-8 TGTAATACGACTCACTATAG NNNNANNNN TCCCACTCAGGCTGGTGTCGGC  67 hTRBV7-1 TGTAATACGACTCACTATAG NNNNANNNN CTCTGAAGTTCCAGCGCACACA  68 hTRBV7-2 TGTAATACGACTCACTATAG NNNNANNNN GATCCAGCGCACACAGCAGGAG  69 hTRBV7-3 TGTAATACGACTCACTATAG NNNNANNNN ACTCTGAAGATCCAGCGCACAGA  70 hTRBV7-5 TGTAATACGACTCACTATAG NNNNANNNN AGATCCAGCGCACAGAGCAAGG  71 hTRBV7-6 TGTAATACGACTCACTATAG NNNNANNNN CAGCGCACAGAGCAGCGGGACT  72 hTRBV7-9 TGTAATACGACTCACTATAG NNNNANNNN GAGATCCAGCGCACAGAGCAGG  73 hTRBV8-1 TGTAATACGACTCACTATAG NNNNANNNN CCCTCAACCCTGGAGTCTACTA  74 hTRBV8-2 TGTAATACGACTCACTATAG NNNNANNNN TCCCCAATCCTGGCATCCACCA  75 hTRBV9 TGTAATACGACTCACTATAG NNNNANNNN CTAAACCTGAGCTCTCTGGAGCT  76 hTRBV10-1 TGTAATACGACTCACTATAG NNNNANNNN CCCTCACTCTGGAGTCTGCTGC  77 hTRBV10-2 TGTAATACGACTCACTATAG NNNNANNNN CCCTCACTCTGGAGTCAGCTAC  78 hTRBV10-3 TGTAATACGACTCACTATAG NNNNANNNN TCCTCACTCTGGAGTCCGCTAC  79 hTRBV11-1 TGTAATACGACTCACTATAG NNNNANNNN CCACTCTCAAGATCCAGCCTGCA  80 hTRBV12-1 TGTAATACGACTCACTATAG NNNNANNNN GAGGATCCAGCCCATGGAACCCA  81 hTRBV12-2 TGTAATACGACTCACTATAG NNNNANNNN CTGAAGATCCAGCCTGCAGAGC  82 hTRBV12-3 TGTAATACGACTCACTATAG NNNNANNNN CAGCCCTCAGAACCCAGGGACT  83 hTRBV13 TGTAATACGACTCACTATAG NNNNANNNN GAGCTCCTTGGAGCTGGGGGACT  84 hTRBV14 TGTAATACGACTCACTATAG NNNNANNNN GGTGCAGCCTGCAGAACTGGAG  85 hTRBV15 TGTAATACGACTCACTATAG NNNNANNNN GACATCCGCTCACCAGGCCTGG  86 hTRBV16 TGTAATACGACTCACTATAG NNNNANNNN TGAGATCCAGGCTACGAAGCTT  87 hTRBV17 TGTAATACGACTCACTATAG NNNNANNNN GAAGATCCATCCCGCAGAGCCG  88 hTRBV18 TGTAATACGACTCACTATAG NNNNANNNN GGATCCAGCAGGTAGTGCGAGG  89 hTRBV19 TGTAATACGACTCACTATAG NNNNANNNN CACTGTGACATCGGCCCAAAAG  90 hTRBV20 TGTAATACGACTCACTATAG NNNNANNNN CTGACAGTGACCAGTGCCCATC  91 hTRBV21 TGTAATACGACTCACTATAG NNNNANNNN GAGATCCAGTCCACGGAGTCAG  92 hTRBV22 TGTAATACGACTCACTATAG NNNNANNNN GTGAAGTTGGCCCACACCAGCCA  93 hTRBV23 TGTAATACGACTCACTATAG NNNNANNNN CCTGGCAATCCTGTCCTCAGAA  94 hTRBV24 TGTAATACGACTCACTATAG NNNNANNNN GAGTCTGCCATCCCCAACCAGA  95 hTRBV25 TGTAATACGACTCACTATAG NNNNANNNN GGAGTCTGCCAGGCCCTCACA  96 hTRBV26 TGTAATACGACTCACTATAG NNNNANNNN GAAGTCTGCCAGCACCAACCAG  97 hTRBV27 TGTAATACGACTCACTATAG NNNNANNNN GGAGTCGCCCAGCCCCAACCAG  98 hTRBV28 TGTAATACGACTCACTATAG NNNNANNNN GGAGTCCGCCAGCACCAACCAG  99 hTRBV29 TGTAATACGACTCACTATAG NNNNANNNN GTGAGCAACATGAGCCCTGAAGA 100 hTRBV30 TGTAATACGACTCACTATAG NNNNANNNN GAGTTCTAAGAAGCTCCTTCTCA

TABLE 5 V segments targeted by each primer used for the amplification of TCR β chain V segments. SEQ TCR b chain V ID NO segment name Targeted V segment(s) 51 hTRBV1 hTRBV01 52 hTRBV2 hTRBV02 53 hTRBV3-1 hTRBV03-1, hTRBV03-2 54 hTRBV4-1 hTRBV04-1 55 hTRBV4-2 hTRBV04-2, hTRBV04-3 56 hTRBV5-1 hTRBV05-1 57 hTRBV5-2 hTRBV05-2 58 hTRBV5-3 hTRBV05-3 59 hTRBV5-4 hTRBV05-4, hTRBV05-5, hTRBV05-6, hTRBV05-7, hTRBV05-8 60 hTRBV6-1 hTRBV06-1 61 hTRBV6-2 hTRBV06-2, hTRBV06-3 62 hTRBV6-4 hTRBV06-4 63 hTRBV6-5 hTRBV06-5 64 hTRBV6-6 hTRBV06-6, hTRBV06-9 65 hTRBV6-7 hTRBV06-7 66 hTRBV6-8 hTRBV06-8 67 hTRBV7-1 hTRBV07-1 68 hTRBV7-2 hTRBV07-2, hTRBV07-8 69 hTRBV7-3 hTRBV07-3, hTRBV07-4 70 hTRBV7-5 hTRBV07-5 71 hTRBV7-6 hTRBV07-6, hTRBV07-7 72 hTRBV7-9 hTRBV07-9 73 hTRBV8-1 hTRBV08-1 74 hTRBV8-2 hTRBV08-2 75 hTRBV9 hTRBV09 76 hTRBV10-1 hTRBV10-1 77 hTRBV10-2 hTRBV10-2 78 hTRBV10-3 hTRBV10-3 79 hTRBV11-1 hTRBV11-1, hTRBV11-2, hTRBV11-3 80 hTRBV12-1 hTRBV12-1 81 hTRBV12-2 hTRBV12-2 82 hTRBV12-3 hTRBV12-3, hTRBV12-4, hTRBV12-5 83 hTRBV13 hTRBV13 84 hTRBV14 hTRBV14 85 hTRBV15 hTRBV15 86 hTRBV16 hTRBV16 87 hTRBV17 hTRBV17 88 hTRBV18 hTRBV18 89 hTRBV19 hTRBV19 90 hTRBV20 hTRBV20 91 hTRBV21 hTRBV21 92 hTRBV22 hTRBV22 93 hTRBV23 hTRBV23 94 hTRBV24 hTRBV24 95 hTRBV25 hTRBV25 96 hTRBV26 hTRBV26 97 hTRBV27 hTRBV27 98 hTRBV28 hTRBV28 99 hTRBV29 hTRBV29 100 hTRBV30 hTRBV30

TABLE 6 Primers for TCR gene amplification. Primer pair for sequencing of TCR α genes: SEQ ID NO 101 and 102. Primer pair for sequencing of TCR β genes: SEQ ID NO 101 and 103. SEQ ID NO Primer name Primer sequence TCR chain 101 Forward primer T7 TRAV/TRBV TGTAATACGACTCACTATAG α and β 102 Reverse primer PCR 1 TRAV GGCCACAGCACTGTTGCTCTTGAAG α 103 Reverse primer PCR 1 TRABV CCACTGTGCACCTCCTTCCCATTC β

TABLE 7 Reverse primers for TCR gene amplification that did not result in successful amplification of PCR products. SEQ ID NO Primer sequence TCR chain 104 TCGACCAGCTTGACATCACAGG α 105 CAGATTTGTTGCTCCAGGCCACAG α 106 TCTGTGATATACACATCAGAATC α 107 GAATCAAAATCGGTGAATAGGCAG α 108 GGCAGACAGACTTGTCACTGGATT α 109 TAGGACACCGAGGTAAAGCCAC β 110 CTGGGTGACGGGTTTGGCCCTAT β 111 TTGACAGCGGAAGTGGTTGC β 112 GGCTGCTCAGGCAGTATCTGGAGTC β 113 GCCAGGCACACCAGTGTGGCCTTTT β

TABLE 8 Primers for addition of Next Generation Sequencing adapters. The primer portion corresponding to the ILLUMINA ® adapters (forward and reverse) is underlined in forward and reverse primers shown below. Primer pair for sequencing of TCR α genes: SEQ ID NOS: 114 and 115. Primer pair for sequencing of TCR β genes: SEQ ID NOS: 114 and 116. SEQ TCR ID NO Primer name Primer sequence chain 114 Forward primer Illumina_T7 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC α and β TRAV/TRBV TCTTCCGATCTTGTAATACGACTCACTATAG 115 Reverse primer PCR 2 TRAC CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGAC α GTGTGCTCTTCCGATCCTCAGCTGGTACACGGCAGGGTCA 116 Reverse primer PCR 2 TRBC CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGAC β GTGTGCTCTTCCGATCAAACACAGCGACCTCGGGTGGGAAC

Example 2: Exemplary Protocol for the SEQTR Method Using Nextera Adapters

TCR α and β chain genes were sequenced in two independent reactions.

1) Starting material and RNA extraction

-   -   To obtain sufficient amounts of RNA in the extraction, a minimum         of 500,000 T-cells were used as starting material.         Alternatively, and especially in instances where fewer T-cells         were available, T-cells were mixed with 50,000 mouse 3T3 cells         that served as carrier. T-cell RNA was extracted using the         RNeasy® Micro Kit from Qiagen Inc. according the manufacturer's         instruction with the following modification: Elution was         performed with 20 μl of water preheated to 50° C. RNA quality         and quantity was verified using a fragment analyzer.

2) cRNA synthesis by in vitro transcription (IVT):

-   -   In vitro transcription of isolated RNA was performed using the         MessageAmp™ II aRNA Amplification Kit from Ambion® (Thermo         Fisher Scientific), which contains enzymes, buffers and         nucleotides required to perform the first and second strand cDNA         and the in vitro transcription. The kit also provides all         columns and reagents needed for the cDNA and cRNA purifications.         RNA amplification was performed according to the manufacturer's         instructions with the following modifications:     -   1) Between 0.5 and 1 μg of total RNA as was used as starting         material. 2) The IVT was performed in a final volume of 40 μl,         and incubated at 37° C. for 16 h. Purified cRNA was quantified         by absorbance using a NanoDrop™ spectrophotometer (Thermo Fisher         Scientific).

3) cDNA synthesis by reverse transcription:

-   -   The reverse transcription of the cRNA was performed with the         SuperScript® III from Invitrogen (Thermo Fisher Scientific). The         kit provides the enzyme, the buffer and the dithiothreitol (DTT)         needed for the reaction. Deoxynucleotides (dNTPs) and RNAsin®         Ribonuclease inhibitor were purchased from Promega. The         sequences for the primers used for the reverse transcription can         be found in Table 9 (primers for sequencing TCR α chain genes)         and Table 10 (primers for sequencing TCR β chain genes).     -   500 ng of cRNA were used as starting material for the reverse         transcription. cRNA was mixed with 1 μl hTRAV or hTRBV primers         mix (2 μM each) and 1 μl dNTP (25 mM) in a final volume of 13         μl. The mix was first incubated at 70° C. for 10 min, then at         50° C. for 30 s. 4 μl 5× buffer, 1 μl DTT (100 mM), 1 μl         SuperScript III and 1 μl RNAsin® were added to the mix. The         samples were subsequently incubated for at 55° C. 1 h and then         at 85° C. for 5 min. After the cDNA synthesis, 1 μg DNase-free         RNase (Roche) was added to the cDNA and incubated at 37° C. for         30 min to remove the cRNA.

4) TCR gene amplification:

-   -   TCR gene amplification was performed using a Phusion®         High-Fidelity DNA polymerase (New England Biolabs) under the         following conditions:     -   PCR mix: 1 μl cDNA from step 3, 0.4 μl dNTPs (10 mM), 0.4 μl         primer mix (20 μM Nextera 5′, 10 μM Reverse primer PCR 1 TRAV or         2.5 μM Reverse primer PCR1 TRBV, see Table 11), 2 μl 5× buffer         and 0.2 μl Phusion® enzyme in a total volume of 10 μl.     -   PCR conditions:         -   94° C. for 5 min         -   20 cycles of             -   98° C. for 10 s             -   55° C. for 30 s             -   72° C. for 30 s         -   72° C. for 2 min     -   PCR products were purified using 1 μl of ExoSAP-IT® PCR Product         Cleanup Kit (Affymetrix) according to the manufacturer's         instructions.

5) Addition of Next Generation Sequencing adapters:

-   -   ILLUMINA® sequencing adapters were added by PCR using a Phusion®         High-Fidelity DNA polymerase (New England Biolabs). The         following mix was added to the 11 μl of PCR1: 1 μl dNTPs (10         mM), 1 μl primer mix (1.25 μM each, see Table 12), 3 μl 5×         buffer and 0.2 μl Phusion® enzyme and 9.8 μl of H₂O.     -   PCR conditions:         -   94° C. for 5 min         -   perform 25 cycles of:             -   98° C. for 10 s             -   55° C. for 30 s             -   72° C. for 30 s         -   72° C. for 2 min

6) TCR library purification:

-   -   10 μl of the PCR product from step 5 were purified using an         AMPURE XP beads (Beckman Coulter) according to the         manufacturer's instruction. Samples could then directly be used         for ILLUMINA® sequencing.

TABLE 9 Preferred primer sequences for amplification of TCR α chain V segments. N can be any nucleotide. The sequences for primers presented in this table consist of three parts (listed from 5′ to 3′): T7 adapter, barcode and TCR α chain V segment. Sequence SEQ Primer name Sequence Nextera adapter barcode portion Sequence TCR α chain V segment ID NO portion of the primer of the primer portion of the primer 261 hTRAV1-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTCTACAGGAGCTCCAGATGAAAG GTATAAGAGACAG 262 hTRAV1-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTTTGAAGGAGCTCCAGATGAAAG GTATAAGAGACAG 263 hTRAV2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TGCTCATCCTCCAGGTGCGGGA GTATAAGAGACAG 264 hTRAV3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAAGAAACCATCTGCCCTTGTGA GTATAAGAGACAG 265 hTRAV4 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTGCCCCGGGTTTCCCTGAGCGAC GTATAAGAGACAG 266 hTRAV5 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCTCTGCGCATTGCAGACACCCA GTATAAGAGACAG 267 hTRAV6 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTGTTTCATATCACAGCCTCCCA GTATAAGAGACAG 268 hTRAV7 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GCTTGTACATTACAGCCGTGCA GTATAAGAGACAG 269 hTRAV8-1/8-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ATCTGAGGAAACCCTCTGTGCA GTATAAGAGACAG 270 hTRAV8-2/8-4 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACCTGACGAAACCCTCAGCCCAT GTATAAGAGACAG 271 hTRAV8-5 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTATGCCTGTCTTTACTTTAATC GTATAAGAGACAG 272 hTRAV8-6 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTGAGGAAACCCTCAGTCCATAT GTATAAGAGACAG 273 hTRAV8-7 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAAACCATCAACCCATGTGAGTGA GTATAAGAGACAG 274 hTRAV9-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACTTGGAGAAAGACTCAGTTCAA GTATAAGAGACAG 275 hTRAV9-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACTTGGAGAAAGGCTCAGTTCAA GTATAAGAGACAG 276 hTRAV10 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTGCACATCACAGCCTCCCA GTATAAGAGACAG 277 hTRAV11 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTTTGGAATATCGCAGCCTCTCAT GTATAAGAGACAG 278 hTRAV12-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTGCTCATCAGAGACTCCAAG GTATAAGAGACAG 279 hTRAV12-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTCTGCTCATCAGAGACTCCCAG GTATAAGAGACAG 280 hTRAV12-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTTGTTCATCAGAGACTCACAG GTATAAGAGACAG 281 hTRAV13-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCTGCACATCACAGAGACCCAA GTATAAGAGACAG 282 hTRAV13-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCTCTGCAAATTGCAGCTACTCAA GTATAAGAGACAG 283 hTRAV14 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTGTCATCTCCGCTTCACAACTGG GTATAAGAGACAG 284 hTRAV15 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTTTTGAATATGCTGGTCTCTCAT GTATAAGAGACAG 285 hTRAV16 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTGAAGAAACCATTTGCTCAAGA GTATAAGAGACAG 286 hTRAV17 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCTTGTTGATCACGGCTTCCCGG GTATAAGAGACAG 287 hTRAV18 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACCTGGAGAAGCCCTCGGTGCA GTATAAGAGACAG 288 hTRAV19 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CACCATCACAGCCTCACAAGTCGT GTATAAGAGACAG 289 hTRAV20 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTTCTGCACATCACAGCCCCTA GTATAAGAGACAG 290 hTRAV21 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTTATACATTGCAGCTTCTCAGCC GTATAAGAGACAG 291 hTRAV22 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTACATTTCCTCTTCCCAGACCAC GTATAAGAGACAG 292 hTRAV23 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CATTGCATATCATGGATTCCCAGC GTATAAGAGACAG 293 hTRAV24 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GCTATTTGTACATCAAAGGATCCC GTATAAGAGACAG 294 hTRAV25 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CAGCTCCCTGCACATCACAGCCA GTATAAGAGACAG 295 hTRAV26-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTGATCCTGCCCCACGCTACGCTGA GTATAAGAGACAG 296 hTRAV26-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTGATCCTGCACCGTGCTACCTTGA GTATAAGAGACAG 297 hTRAV27 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTTCTCTCCACATCACTGCAGCC GTATAAGAGACAG 298 hTRAV28 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GCCACCTATACATCAGATTCCCA GTATAAGAGACAG 299 hTRAV29 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCTCTGCACATTGTGCCCTCCCA GTATAAGAGACAG 300 hTRAV30 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTGTACCTTACGGCCTCCCAGCT GTATAAGAGACAG 301 hTRAV31 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTATCATATCATCATCACAGCCA GTATAAGAGACAG 302 hTRAV32 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCTGCATATTACAGCCACCCAA GTATAAGAGACAG 303 hTRAV33 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACCTCACCATCAATTCCTTAAAAC GTATAAGAGACAG 304 hTRAV34 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCTGCATATCACAGCCTCCCAG GTATAAGAGACAG 305 hTRAV35 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTTCCTGAATATCTCAGCATCCAT GTATAAGAGACAG 306 hTRAV36 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCTGAACATCACAGCCACCCAG GTATAAGAGACAG 307 hTRAV37 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCTGCACATACAGGATTCCCAG GTATAAGAGACAG 308 hTRAV38 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CAAGATCTCAGACTCACAGCTGG GTATAAGAGACAG 309 hTRAV39 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCGTCTCAGCACCCTCCACATCA GTATAAGAGACAG 310 hTRAV40 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCATTGTGAAATATTCAGTCCAGG GTATAAGAGACAG

TABLE 10 Preferred primer sequences for amplification of TCR β chain V segments. N can be any nucleotide. The sequences for primers presented in this table consist of three parts (listed from 5′ to 3′): T7 adapter, barcode and TCR β chain V segment. Sequence SEQ Sequence T7 adapter barcode portion Sequence TCR β chain V segment ID NO Primer name portion of the primer of the primer portion of the primer 311 hTRBV1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTGGTCGCACTGCAGCAAGAAGA GTATAAGAGACAG 312 hTRBV2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GATCCGGTCCACAAAGCTGGAGGA GTATAAGAGACAG 313 hTRBV3-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CATCAATTCCCTGGAGCTTGGTGA GTATAAGAGACAG 314 hTRBV4-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTCACCTACACGCCCTGCAGCCAG GTATAAGAGACAG 315 hTRBV4-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TTCACCTACACACCCTGCAGCCAG GTATAAGAGACAG 316 hTRBV5-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAATGTGAGCACCTTGGAGCTGG GTATAAGAGACAG 317 hTRBV5-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TACTGAGTCAAACACGGAGCTAGG GTATAAGAGACAG 318 hTRBV5-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GCTCTGAGATGAATGTGAGTGCCT GTATAAGAGACAG 319 hTRBV5-4 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTGAGCTGAATGTGAACGCCTT GTATAAGAGACAG 320 hTRBV6-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGTTCTCGCTCAGGCTGGAGT GTATAAGAGACAG 321 hTRBV6-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTGGGGTTGGAGTCGGCTGCTC GTATAAGAGACAG 322 hTRBV6-4 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCCTCACGTTGGCGTCTGCTG GTATAAGAGACAG 323 hTRBV6-5 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCGCTCAGGCTGCTGTCGGC GTATAAGAGACAG 324 hTRBV6-6 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GATTTCCCGCTCAGGCTGGAGT GTATAAGAGACAG 325 hTRBV6-7 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCCCTCAAGCTGGAGTCAGCT GTATAAGAGACAG 326 hTRBV6-8 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCACTCAGGCTGGTGTCGGC GTATAAGAGACAG 327 hTRBV7-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTCTGAAGTTCCAGCGCACACA GTATAAGAGACAG 328 hTRBV7-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GATCCAGCGCACACAGCAGGAG GTATAAGAGACAG 329 hTRBV7-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN ACTCTGAAGATCCAGCGCACAGA GTATAAGAGACAG 330 hTRBV7-5 TCGTCGGCAGCGTCAGATGT HHHHHNNNN AGATCCAGCGCACAGAGCAAGG GTATAAGAGACAG 331 hTRBV7-6 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CAGCGCACAGAGCAGCGGGACT GTATAAGAGACAG 332 hTRBV7-9 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGATCCAGCGCACAGAGCAGG GTATAAGAGACAG 333 hTRBV8-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTCAACCCTGGAGTCTACTA GTATAAGAGACAG 334 hTRBV8-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCCCAATCCTGGCATCCACCA GTATAAGAGACAG 335 hTRBV9 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTAAACCTGAGCTCTCTGGAGCT GTATAAGAGACAG 336 hTRBV10-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTCACTCTGGAGTCTGCTGC GTATAAGAGACAG 337 hTRBV10-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCCTCACTCTGGAGTCAGCTAC GTATAAGAGACAG 338 hTRBV10-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TCCTCACTCTGGAGTCCGCTAC GTATAAGAGACAG 339 hTRBV11-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCACTCTCAAGATCCAGCCTGCA GTATAAGAGACAG 340 hTRBV12-1 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGGATCCAGCCCATGGAACCCA GTATAAGAGACAG 341 hTRBV12-2 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTGAAGATCCAGCCTGCAGAGC GTATAAGAGACAG 342 hTRBV12-3 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CAGCCCTCAGAACCCAGGGACT GTATAAGAGACAG 343 hTRBV13 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGCTCCTTGGAGCTGGGGGACT GTATAAGAGACAG 344 hTRBV14 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGTGCAGCCTGCAGAACTGGAG GTATAAGAGACAG 345 hTRBV15 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GACATCCGCTCACCAGGCCTGG GTATAAGAGACAG 346 hTRBV16 TCGTCGGCAGCGTCAGATGT HHHHHNNNN TGAGATCCAGGCTACGAAGCTT GTATAAGAGACAG 347 hTRBV17 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAAGATCCATCCCGCAGAGCCG GTATAAGAGACAG 348 hTRBV18 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGATCCAGCAGGTAGTGCGAGG GTATAAGAGACAG 349 hTRBV19 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CACTGTGACATCGGCCCAAAAG GTATAAGAGACAG 350 hTRBV20 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CTGACAGTGACCAGTGCCCATC GTATAAGAGACAG 351 hTRBV21 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGATCCAGTCCACGGAGTCAG GTATAAGAGACAG 352 hTRBV22 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTGAAGTTGGCCCACACCAGCCA GTATAAGAGACAG 353 hTRBV23 TCGTCGGCAGCGTCAGATGT HHHHHNNNN CCTGGCAATCCTGTCCTCAGAA GTATAAGAGACAG 354 hTRBV24 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGTCTGCCATCCCCAACCAGA GTATAAGAGACAG 355 hTRBV25 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGAGTCTGCCAGGCCCTCACA GTATAAGAGACAG 356 hTRBV26 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAAGTCTGCCAGCACCAACCAG GTATAAGAGACAG 357 hTRBV27 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGAGTCGCCCAGCCCCAACCAG GTATAAGAGACAG 358 hTRBV28 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GGAGTCCGCCAGCACCAACCAG GTATAAGAGACAG 359 hTRBV29 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GTGAGCAACATGAGCCCTGAAGA GTATAAGAGACAG 360 hTRBV30 TCGTCGGCAGCGTCAGATGT HHHHHNNNN GAGTTCTAAGAAGCTCCTTCTCA GTATAAGAGACAG

TABLE 11 Primers for TCR gene amplification. Primer pair for sequencing of TCR α genes: SEQ ID NOs: 256 and 257. Primer pair for sequencing of TCR β genes: SEQ ID NOs: 256 and 258. The primer portion corresponding to the ILLUMINA ® adapters (forward and reverse) is underlined in reverse primers shown below. SEQ ID NO Primer name Primer sequence TCR chain 256 Forward primer Nextera 5′ TCGTCGGCAGCGTC α and β 257 Reverse primer PCR 1 TRAV GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG α GAATCAAAATCGGTGAATAGGCAG 258 Reverse primer PCR 1 TRBV GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG β GCCAGGCACACCAGTGTGGCCTTTT

TABLE 12 Primers used to add the full Nextera sequence to both TCRα and TCRβ. SEQ ID NO Primer name Primer sequence TCR chain 259 Index Read 1 CAAGCAGAAGACGGCATACGAGAT [i7] GTCTCGTGGGCTC α and β 260 Index Read 2 AATGATACGGCGACCACCGAGATCTACAC [i5] TCGTCGGCAGCG α

Example 3: Exemplary Protocol for the SEQTR Method without In Vitro Transcription

TCR α and β chain genes were sequenced in two independent reactions.

1) Starting material and RNA extraction

-   -   To obtain sufficient amounts of RNA in the extraction, a minimum         of 500,000 T-cells were used as starting material.         Alternatively, and especially in instances where fewer T-cells         were available, T-cells were mixed with 50,000 mouse 3T3 cells         that served as carrier. T-cell RNA was extracted using the         RNeasy® Micro Kit from Qiagen Inc. according the manufacturer's         instruction with the following modification: Elution was         performed with 20 μl of water preheated to 50° C. RNA quality         and quantity was verified using a fragment analyzer.

2) cDNA synthesis by reverse transcription:

-   -   The reverse transcription of the RNA was performed with the         SuperScript® III from Invitrogen (Thermo Fisher Scientific) and         oligo d(T). The kit provides the enzyme, the buffer and the         dithiothreitol (DTT) needed for the reaction. Deoxynucleotides         (dNTPs), oligo d(T) and RNAsin® Ribonuclease inhibitor were         purchased from Promega.     -   500 ng of RNA were used as starting material for the reverse         transcription. RNA was mixed with 1 μl of oligo d(T) and 1 μl         dNTP (25 mM) in a final volume of 13 μl. The mix was first         incubated at 70° C. for 10 min, then at 50° C. for 30 s. 4 μl 5         × buffer, 1 μl DTT (100 mM), 1 μl SuperScript III and 1 μl         RNAsin® were added to the mix. The samples were subsequently         incubated for at 55° C. 1 h and then at 85° C. for 5 min.

3) Second strand cDNA synthesis:

-   -   cDNA was then used to synthesize the second strand, performed         using the Phusion® High-Fidelity DNA polymerase (New England         Biolabs) under the following conditions:     -   Mix: 20 μl cDNA from step 2, 4 μl dNTPs (10 mM), 2 μl TRAV         primer mix (Table 9), 2 μl TRBV primers mix (Table 10), 20 μl 5×         buffer, 1 μl Phusion® enzyme in a total volume of 100 μl.     -   Synthesis conditions:         -   98° C. for 5 min         -   40° C. for 30 s         -   72° C. for 5 min

4) cDNA purification:

-   -   100 μl of the cDNA product from step 3 were purified using an         AMPURE XP beads (Beckman Coulter) according to the         manufacturer's instruction.

5) TCR gene amplification:

-   -   TCR gene amplification was performed using a Phusion®         High-Fidelity DNA polymerase (New England Biolabs) under the         following conditions: PCR mix: 7 μl cDNA from step 4, 0.4 μl         dNTPs (10 mM), 0.4 μl primer mix (20 μM Nextera5′, 10 μM Reverse         primer PCR 1 TRAV or 2.5 μM Reverse primer PCR1 TRBV, see Table         11), 2 μl 5× buffer and 0.2 μl Phusion® enzyme in a total volume         of 10 μl.     -   PCR conditions:         -   94° C. for 5 min         -   20 cycles of             -   98° C. for 10 s             -   55° C. for 30 s             -   72° C. for 30 s         -   72° C. for 2 min     -   PCR products were purified using 1 μl of ExoSAP-IT® PCR Product         Cleanup Kit (Affymetrix) according to the manufacturer's         instructions.

6) Addition of Next Generation Sequencing adapters:

-   -   ILLUMINA® sequencing adapters were added by PCR using a Phusion®         High-Fidelity DNA polymerase (New England Biolabs). The         following mix was added to the 11 μl of PCR1: 1 μl dNTPs (10         mM), 1 μl primer mix (1.25 μM each, see Table 12), 3 μl 5×         buffer and 0.2 μl Phusion® enzyme and 9.8 μl of H₂O.     -   PCR conditions:         -   94° C. for 5 min         -   perform 25 cycles of:             -   98° C. for 10 s             -   55° C. for 30 s             -   72° C. for 30 s         -   72° C. for 2 min

7) TCR library purification:

-   -   10 μl of the PCR product from step 5 were purified using an         AMPURE XP beads (Beckman Coulter) according to the         manufacturer's instruction. Samples could then directly be used         for ILLUMINA® sequencing.

Example 4: Sensitivity of the TCR Sequencing Method

One of the challenges of TCR sequencing are the small amounts of genetic material for each T-cell clone. In many cases, the number of T-cells that can be recovered from a given experiment is too small for researchers to directly extract sufficient amounts of RNA for a subsequent amplification of the TCR genes. In such instances, the T-cells of interest can be mixed with 3T3 mouse cells, which serve as a carrier.

5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively. The RNA of each mixture was isolated and subjected to steps 2 to 4 of the SEQTR method outlined above (see Detailed Description of the Invention). PCR products were separated on an agarose gel and visualized.

No TCR-specific PCR products were observed in samples that only contained 3T3 cells (see FIG. 4 ). However, TCR-specific bands were detected in all other samples: Increasing amounts of CD8 positive T-cells in the samples were correlated with increasing amounts of TCR-specific PCR products and decreasing intensity of the unspecific dimer primer band. These data demonstrate that the SEQTR method is sensitive enough to amplify TCR genes from as little as 1,000 T-cells, with no detectable background signal from the 3T3 carrier cells.

Example 5: Specificity of the SEQTR Method

Another challenge of TCR sequencing is the lack of specific amplification of TCR genes from complex samples. Competing TCR sequencing technologies such as services offered by Adaptive Biotechnology are characterized by up to 90% unspecific amplification. As a result, only as little as 10% of all sequencing data are informative for TCR repertoire determination, increasing cost and duration of any project aiming to sequence TCR repertoires.

5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively. TCR repertoires for the individual samples were sequenced using the SEQTR method, and the percentage of reads that corresponded to TCR or non-TCR sequences, respectively, was determined. As shown in FIG. 5 , 93-97% of all sequencing reads indeed corresponded to TCR genes, independent of the amount of T-cells used as starting material. In summary, these data show that TCR amplification using the SEQTR method is highly specific even when as little as 1,000 T-cells are used as starting material.

Example 6: Unambiguous Identification of TCR Genes

In humans, the TCR locus comprises 54 different V segments for the TCR α chain and 65 different V segments for the TCR β chain. However, many of these V segments are highly homologous. Consequently, one of the big challenges of TCR sequencing is to successfully differentiate between two or more TCR gene segments with high degrees of homology. For instance, depending on the choice of primer used in the amplification of the TCR gene and the length of the generated PCR product, the resulting sequencing data might be compatible with more than one V or J segment (in other words, two or more TCR V or J segments show 100% homology in the sequenced region). In these cases, the TCR gene for a specific read cannot be unambiguously assigned/identified.

5×10{circumflex over ( )}4 3T3 cells were mixed with 10{circumflex over ( )}6, 10{circumflex over ( )}5, 10{circumflex over ( )}4, 10{circumflex over ( )}3 or 0 CD8 positive T-cells, respectively. The RNA of each mixture was isolated and subjected to the TCR sequencing method. Out of all the sequencing reads that were identified as TCR genes, it was assessed if the V or J segments could be identified unambiguously. The data show that between 95% and 97% of all TCR sequencing reads could be assigned to a specific TCR segment, even when using as little as 1,000 T-cells as genetic starting material (see FIG. 6 ). In summary, the data demonstrate the robustness of the SEQTR method as 90 to 93% of all reads can be used to identify TCR sequences once unspecific sequences and ambiguous TCR sequences have been removed.

Due to the homology between V segments, it can be sometimes difficult to clearly identify the TCR sequence. hTRBV6-2 and hTRBV6-3 cannot be differentiated as they have 100% homology and thus will code for the same TCR. Due to their sequences, hTRBV12-3 and hTRBV12-4 cannot be differentiated with the method disclosed herein. Only paired-end sequencing that will catch the 5′-end of the V segment can discriminate these two sequences. Thus the hTRBV12-3 and hTRBV12-4 were considered as a unique sequence for the analysis of the repertoire.

Example 7: Linearity of TCR Gene Amplification

Because non-linear amplification of individual TCR sequences can lead to an incorrect over- or underrepresentation of the affected TCR genes in the final TCR repertoire, linearity of amplification is a critical determinant of the reliability and quality of the TCR sequencing data.

To test linearity of TCR gene amplification in our system, a fixed amount of DNA encoding a known TCR sequence was diluted at different concentrations into a DNA pool representing a naïve CD8 repertoire. Subsequently, the TCR repertoire of each sample was analyzed with SEQTR.

The observed frequency of the known TCR sequence in the entire TCR repertoire was then sequenced for each dilution and compared to the expected frequency. The scatter plot in FIG. 7 shows an excellent correlation (R²=0.99) between the dilution and the frequency of the known TCR sequence in the repertoire observed after sequencing. These data confirm the linearity of the amplification and suggest that results obtained using the SEQTR technique are quantitative.

Example 8: Reproducibility of the SEQTR Method

The reproducibility of the method was tested by performing two independent technical replicates starting from the same sample. The frequencies for each V-J rearrangement in the TCR β chains were determined and compared between the two replicates, as illustrated in FIG. 8 . Each sphere represents a single V-J rearrangement that was detected in both replicates. Each sphere represents a single V-J rearrangement with the size of a sphere indicating the relative frequency of the specific V-J recombination. Grey spheres represent rearrangements for which the relative frequencies detected in the two replicates differed by less than two-fold. Black spheres represent rearrangements for which the relative frequencies detected in the two replicates differed by more than two-fold. Consistent with common practice in the analysis of gene expression data, differences between replaces of less than 2-fold are not considered significant.

The data show that only 13% of all V-J rearrangements showed a significant frequency difference of more than two-fold between the two technical replicates (see FIG. 8 upper inset). However, as illustrated in FIG. 8 , V-J recombinations that were significantly different between the technical replicates were rather poorly expressed, as indicated by the small sizes of the black spheres. Therefore, if the frequencies of the individual V-J rearrangement are taken into consideration, only 0.5% of the sequences showed more than a two-fold difference between the replicates (see FIG. 8 lower inset), demonstrating that the SEQTR method is very reproducible.

Example 9: Sequencing of Example Repertoires Using the SEQTR Method

The SEQTR method was tested on three different type of CD8 positive T-cells:

-   -   (1) T-cell population 1: CD8 positive T-cells isolated from         peripheral blood mononuclear cells (PBMCs).     -   (2) T-cell population 2: CD8 positive T-cells as in population 1         were FACS sorted using tetramers. Tetramers are MHC molecules         presenting a specific peptide, linked to fluorescent dye.         Tetramers bind T-cell expressing a TCR that specifically         recognizes the peptide. The fluorescent dye allows sorting of         the the desired T-cells by FACS.     -   (3) T-cell population 3: CD8 positive T-cells as in population 2         that were subsequently expanded in vitro.

The relative frequencies of each V-J rearrangement were determined using the SEQTR method (see FIG. 9 ). As expected, the naïve TCR repertoire derived from PBMC (population 1) is highly diverse (see FIG. 9A). Almost all the possible V-J rearrangements are represented in the sample, with no single V-J rearrangement exhibiting a frequency of over 11% The repertoire of the tetramer sorted CD8 positive T-cell subset 2 (see FIG. 9B) is less diverse as compared to the naïve one. Not only are fewer V-J rearrangements present in the repertoire overall. Moreover, two V-J rearrangements are clearly dominant, exhibiting frequencies of over 20%. These V-J rearrangements represent the few T-cells that recognize the epitope TEDYMIHII (SEQ ID NO: 236) conjugated to the tetramer and that were enriched during the tetramer purification step. Finally, the rapid clonal expansion (population 3) of the tetramer-purified T-cells enhances the bias of the TCR repertoire towards the T-cell clones already dominating subset 2. Consequently, part of the low frequency V-J rearrangements are lost and not detected anymore (see FIG. 9C). In summary, these data illustrate that the SEQTR method is well suited to differentiate between TCR repertoires with different degrees of diversities.

Example 10: Comparison of the SEQTR Method with Low-Throughput Single Cell Cloning

In order to determine how accurate the TCR repertoire data obtained using the SEQTR method were as compared to the true TCR repertoire present in a given T-cell population, we compared our results to data obtained by single cell sequencing.

Tetramer-specific CD8 were sorted from PBMC by FACS. The recovered cell population was split in two. Half of the cells were subjected to the SEQTR method to sequence the TCR repertoire. For the other half of the cells, individual T-cell clones were isolated and expanded in vitro (single cell cloning). Once the clones were established, the TCR genes of each T-cell clones were amplified and sequenced using classical Sanger sequencing (see FIG. 10A).

Among the 42 individual clones tested using the single cell method, six different TCRs were identified (see FIG. 10B). Using the SEQTR method, 116 different TCR genes were found (the eight most frequently observed V-J rearrangements are shown in FIG. 10C, also see Table 13 and Table 14). Indeed, the five TCRs most frequently observed with the single cell cloning technique also correspond to the five clones most frequently observed when applying the SEQTR method. Overall, all six TCR clones identified with single cell sequencing are represented among the eight TCRs with the highest frequencies observed in the SEQTR method. In summary, these data suggest that the SEQTR method produces a true representation of the actual TCR repertoire of a given T-cell population.

TABLE 13 CDR3 regions of TCR clones identified using the single cell sequencing method. SEQ ID NO CDR3 region 237 CASSRHVGGVPEAFFG 238 CASSIGRGSEQYFG 239 CASSDVLSGEAFFG 240 CASQGHKNTEAFFG 241 CASSLGPGGVKTNEKLFFG 242 CASSLGPGGVKTNEKLFFG

TABLE 14 Eight most frequently observed V-J rearrangements of the 116 different TCR genes identified using the SEQTR method. SEQ ID NO CDR3 region 243 CASSDVLSGEAFFG 244 CASQGHKNTEAFFG 245 CASSLGPGGVKTNEKLFFG 246 CASSIGRGSEQYFG 247 CASSRHVGGVPEAFFG 248 CASSASKGQPQHFG 249 CASQGHKNTEAFFG 250 CASSLGPGGVKTNEKLFFG

Example 11: TCR Sequencing Services Offered by Adaptive Biotechnology Provide Sequencing Data that May Reflect Up to 90% Unspecific TCR Amplification

Tumor samples from 16 patients were collected and the tumor cells were separated from the surrounding tissue (stroma). In addition, epitope-specific TIL were sorted by FACS from the tumor samples using tetramer staining (TET). Finally, the tumor cells were engrafted into humanized mice. After some time, the tumor was collected and epitope specific TIL were sorted by FACS.

DNA extraction was performed for each sample. DNA was sent to Adaptive Biotechnology for TCR sequencing (immunoSEQ® method, survey protocol 200,000-300,000 reads per sample).

In 80% of the samples, the immunoSEQ® method failed to generate 200,000 reads per samples, suggesting that the immunoSEQ® method fails to generate TCR repertoires with significant reliability.

Example 12: Amplification of TCR Genes from PBMC and CD4 Positive T-Cells

RNA was isolated from 10{circumflex over ( )}6 PBMC or 10{circumflex over ( )}6 CD4 positive T-cells, respectively, from three independent samples, The RNA was then subjected to steps 2 to 4 of the SEQTR method outlined above (see Detailed Description of the Invention). PCR products were separated on an agarose gel and visualized (see FIG. 12 ). Only TCR-specific bands are observed, suggesting that the SEQTR method cannot only be used for CD8 positive T-cells (see Example 7), but also for CD4 positive T-cells and even for T-cells that are part of a complex mixture of other PBMCs.

The foregoing examples and description of the embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference herein in their entireties.

As described and claimed herein, including in the accompanying drawings, reference is made to particular features, including method steps. It is to be understood that the disclosure of the invention in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment, or a particular claim, that feature can also be used, to the extent possible, in combination with and/or in the context of other particular aspects and embodiments, and in the disclosed methods, systems and kits generally.

Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes that possibility).

TABLE 15 TCR α chain V segments and binding sites for primers presented in Table 2 and Table 9. The sequence for each V segment presented in this table consists of three parts (listed from 5′ to 3′): Sequence upstream of primer binding site, sequence of the primer binding site, sequence downstream of the primer binding site. hTRAV Primer sequence binding downstream V site of primer segment Primer SEQ within binding name name ID NO hTRAV sequence upstream of primer binding site hTRAV site hTRA hTRA 117 ATGTGGGGAGCTTTCCTTCTCTATGTTTCCATGAAGATGGGAGGCACT CTTCT ACTCTGC V01-1 V01-1 GCAGGACAAAGCCTTGAGCAGCCCTCTGAAGTGACAGCTGTGGAAGGA ACAGG CTCTTAC GCCATTGTCCAGATAAACTGCACGTACCAGACATCTGGGTTTTATGGG AGCTC TTCTGCG CTGTCCTGGTACCAGCAACATGATGGCGGAGCACCCACATTTCTTTCT CAGAT CTGTGAG TACAATGCTCTGGATGGTTTGGAGGAGACAGGTCGTTTTTCTTCATTC GAAAG AGA CTTAGTCGCTCTGATAGTTATGGTTACCTC hTRA hTRA 118 ATGTGGGGAGTTTTCCTTCTTTATGTTTCCATGAAGATGGGAGGCACT CTTTT ACTCTGC V01-2 V0 1-2 ACAGGACAAAACATTGACCAGCCCACTGAGATGACAGCTACGGAAGGT GAAGG CTCTTAC GCCATTGTCCAGATCAACTGCACGTACCAGACATCTGGGTTCAACGGG AGCTC CTCTGTG CTGTTCTGGTACCAGCAACATGCTGGCGAAGCACCCACATTTCTGTCT CAGAT CTGTGAG TACAATGTTCTGGATGGTTTGGAGGAGAAAGGTCGTTTTTCTTCATTC GAAAG AGA CTTAGTCGGTCTAAAGGGTACAGTTACCTC hTRA hTRA 119 ATGGCTTTGCAGAGCACTCTGGGGGCGGTGTGGCTAGGGCTTCTCCTC TGCTC GGCAGAT V02 V02 AACTCTCTCTGGAAGGTTGCAGAAAGCAAGGACCAAGTGTTTCAGCCT ATCCT GCTGCTG TCCACAGTGGCATCTTCAGAGGGAGCTGTGGTGGAAATCTTCTGTAAT CCAGG TTTACTA CACTCTGTGTCCAATGCTTACAACTTCTTCTGGTACCTTCACTTCCCG TGCGG CTGTGCT GGATGTGCACCAAGACTCCTTGTTAAAGGCTCAAAGCCTTCTCAGCAG GA GTGGAGG GGACGATACAACATGACCTATGAACGGTTCTCTTCATCGC A hTRA hTRA 120 ATGGCCTCTGCACCCATCTCGATGCTTGCGATGCTCTTCACATTGAGT GAAGA GCGACTC V03 V03 GGGCTGAGAGCTCAGTCAGTGGCTCAGCCGGAAGATCAGGTCAACGTT AACCA CGCTTTG GCTGAAGGGAATCCTCTGACTGTGAAATGCACCTATTCAGTCTCTGGA TCTGC TACTTCT AACCCTTATCTTTTTTGGTATGTTCAATACCCCAACCGAGGCCTCCAG CCTTG GTGCTGT TTCCTTCTGAAATACATCACAGGGGATAACCTGGTTAAAGGCAGCTAT TGA GAGAGAC GGCTTTGAAGCTGAATTTAACAAGAGCCAAACCTCCTTCCACCT A hTRA hTRA 121 ATGAGGCAAGTGGCGAGAGTGATCGTGTTCCTGACCCTGAGTACTTTG CCTGC ACTGCTG V04 V04 AGCCTTGCTAAGACCACCCAGCCCATCTCCATGGACTCATATGAAGGA CCCGG TGTACTA CAAGAAGTGAACATAACCTGTAGCCACAACAACATTGCTACAAATGAT GTTTC CTGCCTC TATATCACGTGGTACCAACAGTTTCCCAGCCAAGGACCACGATTTATT CCTGA GTGGGTG ATTCAAGGATACAAGACAAAAGTTACAAACGAAGTGGCCTCCCTGTTT GCGAC ACA ATCCCTGCCGACAGAAAGTCCAGCACTCTGAG hTRA hTRA 122 ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCTG TCTCT GACTGGG V05 V05 GACTGTATGAGTAGAGGAGAGGATGTGGAGCAGAGTCTTTTCCTGAGT GCGCA GACTCAG GTCCGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGC TTGCA CTATCTA TCCTCCACCTACTTATACTGGTATAAGCAAGAACCTGGAGCAGGTCTC GACAC CTTCTGT CAGTTGCTGACGTATATTTTTTCAAATATGGACATGAAACAAGACCAA CCA GCAGAGA AGACTCACTGTTCTATTGAATAAAAAGGATAAACATCTG GTA hTRA hTRA 123 ATGGAGTCATTCCTGGGAGGTGTTTTGCTGATTTTGTGGCTTCAAGTG TTGTT GCCTGCA V06 V06 GACTGGGTGAAGAGCCAAAAGATAGAACAGAATTCCGAGGCCCTGAAC TCATA GACTCAG ATTCAGGAGGGTAAAACGGCCACCCTGACCTGCAACTATACAAACTAT TCACA CTACCTA TCCCCAGCATACTTACAGTGGTACCGACAAGATCCAGGAAGAGGCCCT GCCTC CCTCTGT GTTTTCTTGCTACTCATACGTGAAAATGAGAAAGAAAAAAGGAAAGAA CCA GCTCTAG AGACTGAAGGTCACCTTTGATACCACCCTTAAACAGAGT ACA hTRA hTRA 124 ATGGAGAAGATGCGGAGACCTGTCCTAATTATATTTTGTCTATGTCTT GCTTG GCCTGAA V07 V07 GGCTGGGCAAATGGAGAAAACCAGGTGGAGCACAGCCCTCATTTTCTG TACAT GATTCAG GGACCCCAGCAGGGAGACGTTGCCTCCATGAGCTGCACGTACTCTGTC TACAG CCACCTA AGTCGTTTTAACAATTTGCAGTGGTACAGGCAAAATACAGGGATGGGT CCGTG TTTCTGT CCCAAACACCTATTATCCATGTATTCAGCTGGATATGAGAAGCAGAAA CA GCTGTAG GGAAGACTAAATGCTACATTACTGAAGAATGGAAGCA ATG hTRA hTRA 125 ATGCTCCTGTTGCTCATACCAGTGCTGGGGATGATTTTTGCCCTGAGA ATCTG GTGGAGT V08-1 V08- GATGCCAGAGCCCAGTCTGTGAGCCAGCATAACCACCACGTAATTCTC AGGAA GACACAG 1/08-3 TCTGAAGCAGCCTCACTGGAGTTGGGATGCAACTATTCCTATGGTGGA ACCCT CTGAGTA ACTGTTAATCTCTTCTGGTATGTCCAGTACCCTGGTCAACACCTTCAG CTGTG CTTCTGT CTTCTCCTCAAGTACTTTTCAGGGGATCCACTGGTTAAAGGCATCAAG CA GCCGTGA GGCTTTGAGGCTGAATTTATAAAGAGTAAATTCTCCTTTA ATGC hTRA hTRA 126 ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACTCTGGGA ACCTG ATGAGCG V08-2 V08- GGAACCAGAGCCCAGTCGGTGACCCAGCTTGACAGCCACGTCTCTGTC ACGAA ACGCGGC 2/08-4 TCTGAAGGAACCCCGGTGCTGCTGAGGTGCAACTACTCATCTTCTTAT ACCCT TGAGTAC TCACCATCTCTCTTCTGGTATGTGCAACACCCCAACAAAGGACTCCAG CAGCC TTCTGTG CTTCTCCTGAAGTACACATCAGCGGCCACCCTGGTTAAAGGCATCAAC CAT TTGTGAG GGTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCC TGA hTRA hTRA 127 ATGCTCCTGGAGCTTATCCCACTGCTGGGGATACATTTTGTCCTGAGA ATCTG TTGGAGT V08-3 V08- ACTGCCAGAGCCCAGTCAGTGACCCAGCCTGACATCCACATCACTGTC AGGAA GATGCTG 1/08-3 TCTGAAGGAGCCTCACTGGAGTTGAGATGTAACTATTCCTATGGGGCA ACCCT CTGAGTA ACACCTTATCTCTTCTGGTATGTCCAGTCCCCCGGCCAAGGCCTCCAG CTGTG CTTCTGT CTGCTCCTGAAGTACTTTTCAGGAGACACTCTGGTTCAAGGCATTAAA CA GCTGTGG GGCTTTGAGGCTGAATTTAAGAGGAGTCAATCTTCCTTCA GTGC hTRA hTRA 128 ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACCCTGGGA ACCTG ATGAGCG V08-4 V08- GGAACCAGAGCCCAGTCGGTGACCCAGCTTGGCAGCCACGTCTCTGTC ACGAA ACGCGGC 2/08-4 TCTGAAGGAGCCCTGGTTCTGCTGAGGTGCAACTACTCATCGTCTGTT ACCCT TGAGTAC CCACCATATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAG CAGCC TTCTGTG CTTCTCCTGAAGTACACATCAGCGGCCACCCTGGTTAAAGGCATCAAC CAT CTGTGAG GGTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCC TGACACA hTRA hTRA 129 ATGCTCCTGGTGCTCATCCCACTGCTGGGGATACATTTTGTCCTGAGT CCTAT TCTTAAT V08-5 V08-5 GAGAACTGTCAGAGCCCAGTCAGTGACCCAGCCTGACATCCGCATCAC GCCTG CCTGTCA TGTCTCTGAAGGAGCCTCACTGGAGTTGAGATGTAACTATTCCTATGG TCTTT GCTGAGG GGCGATGTTGTGGGAAGTCAGGGACCCCAAACGGAGGGACCGGCTGAA ACTTT AGGATGT GCCATGGCAGAAGAATGTGGATTGTGAAGATTTCATGGACATTTATTA AATC ATGTCAC GTTCCCCAAATTAATACTTTTATAATTTCTTATGCCTCTCTTTACTGC C AATCTCTAAACATAAATTGTAAAGATTTCATGGACACTTATCACTTCC CCAATCAATACCCCTGTGATTT hTRA hTRA 130 ATGCTCCTGCTGCTCGTCCCAGCGTTCCAGGTGATTTTTACCCTGGGA CTTGA AAGCGAC V08-6 V08-6 GGAACCAGAGCCCAGTCTGTGACCCAGCTTGACAGCCAAGTCCCTGTC GGAAA ACGGCTG TTTGAAGAAGCCCCTGTGGAGCTGAGGTGCAACTACTCATCGTCTGTT CCCTC AGTACTT TCAGTGTATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAG AGTCC CTGTGCT CTTCTCCTGAAGTATTTATCAGGATCCACCCTGGTTAAAGGCATCAAC ATAT GTGAGTG GGTTTTGAGGCTGAATTTAACAAGAGTCAAACTTCCTTCCA A hTRA hTRA 131 ATGCTCTTAGTGGTCATTCTGCTGCTTGGAATGTTCTTCACACTGAGA GAAAC TGCTGCT V08-7 V08-7 ACCAGAACCCAGTCGGTGACCCAGCTTGATGGCCACATCACTGTCTCT CATCA GAGTACT GAAGAAGCCCCTCTGGAACTGAAGTGCAACTATTCCTATAGTGGAGTT ACCCA TCTGTGC CCTTCTCTCTTCTGGTATGTCCAATACTCTAGCCAAAGCCTCCAGCTT TGTGA TGTGGGT CTCCTCAAAGACCTAACAGAGGCCACCCAGGTTAAAGGCATCAGAGGT GTGA GACAGG TTTGAGGCTGAATTTAAGAAGAGCGAAACCTCCTTCTACCTGAG hTRA hTRA 132 ATGAATTCTTCTCCAGGACCAGCGATTGCACTATTCTTAATGTTTGGG ACTTG GAGTCAG V09-1 V09-1 GGAATCAATGGAGATTCAGTGGTCCAGACAGAAGGCCAAGTGCTCCCC GAGAA ACTCCGC TCTGAAGGGGATTCCCTGATTGTGAACTGCTCCTATGAAACCACACAG AGACT TGTGTAC TACCCTTCCCTTTTTTGGTATGTCCAATATCCTGGAGAAGGTCCACAG CAGTT TTCTGTG CTCCACCTGAAAGCCATGAAGGCCAATGACAAGGGAAGGAACAAAGGT CAA CTCTGAG TTTGAAGCCATGTACCGTAAAGAAACCACTTCTTTCC TGA hTRA hTRA 133 ATGAACTATTCTCCAGGCTTAGTATCTCTGATACTCTTACTGCTTGGA ACTTG GTGTCAG V09-2 V09-2 AGAACCCGTGGAAATTCAGTGACCCAGATGGAAGGGCCAGTGACTCTC GAGAA ACTCAGC TCAGAAGAGGCCTTCCTGACTATAAACTGCACGTACACAGCCACAGGA AGGCT GGTGTAC TACCCTTCCCTTTTCTGGTATGTCCAATATCCTGGAGAAGGTCTACAG CAGTT TTCTGTG CTCCTCCTGAAAGCCACGAAGGCTGATGACAAGGGAAGCAACAAAGGT CAA CTCTGAG TTTGAAGCCACATACCGTAAAGAAACCACTTCTTTCC TGA hTRA hTRA 134 ATGAAAAAGCATCTGACGACCTTCTTGGTGATTTTGTGGCTTTATTTT CTGCA GCTCAGC V10 V10 TATAGGGGGAATGGCAAAAACCAAGTGGAGCAGAGTCCTCAGTCCCTG CATCA GATTCAG ATCATCCTGGAGGGAAAGAACTGCACTCTTCAATGCAATTATACAGTG CAGCC CCTCCTA AGCCCCTTCAGCAACTTAAGGTGGTATAAGCAAGATACTGGGAGAGGT TCCCA CATCTGT CCTGTTTCCCTGACAATCATGACTTTCAGTGAGAACACAAAGTCGAAC GTGGTGA GGAAGATATACAGCAACTCTGGATGCAGACACAAAGCAAAGCTCT GCG hTRA hTRA 135 ACGGAGAAGCCCTTGGGAGTTTCATTCTTGATTTCCTCCTGGCAGCTG GTTTG CTGGGAG V11 VII TGCTGGGTGAATAGACTACATACACTGGAGCAGAGTCCTTCATTCCTG GAATA ATTCAGC AATATTCAGGAGGGAATGCATGCCGTTCTTAATTGTACTTATCAGGAG TCGCA CACCTAC AGAACACTCTTCAATTTCCACTGGTTCCGGCAGGATCCGGGGAGAAGA GCCTC TTCTGTG CTTGTGTCTTTGACCTTAATTCAATCAAGCCAGAAGGAGCAGGGAGAC TCAT CTTTGC AAATATTTTAAAGAACTGCTTGGAAAAGAAAAATTTTATAGT hTRA hTRA 136 ATGATATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGC CCCTG CTCAGTG V12-1 V12-1 TGGGTTTGGAGCCAACGGAAGGAGGTGGAGCAGGATCCTGGACCCTTC CTCAT ATTCAGC AATGTTCCAGAGGGAGCCACTGTCGCTTTCAACTGTACTTACAGCAAC CAGAG CACCTAC AGTGCTTCTCAGTCTTTCTTCTGGTACAGACAGGATTGCAGGAAAGAA ACTCC CTCTGTG CCTAAGTTGCTGATGTCCGTATACTCCAGTGGTAATGAAGATGGAAGG AAG TGGTGAA TTTACAGCACAGCTCAATAGAGCCAGCCAGTATATTT CA hTRA hTRA 137 ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGC CTCTG CCCAGTG V12-2 V12-2 TGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTC CTCAT ATTCAGC AGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGAC CAGAG CACCTAC CGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGC ACTCC CTCTGTG CCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGA CAG CCGTGAA AGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTT hTRA hTRA 138 ATGAAATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGC CCTTG CCCAGTG V12-3 V12-3 TGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGGATCCTGGACCACTC TTCAT ATTCAGC AGTGTTCCAGAGGGAGCCATTGTTTCTCTCAACTGCACTTACAGCAAC CAGAG CACCTAC AGTGCTTTTCAATACTTCATGTGGTACAGACAGTATTCCAGAAAAGGC ACTCA CTCTGTG CCTGAGTTGCTGATGTACACATACTCCAGTGGTAACAAAGAAGATGGA CAG CAATGAG AGGTTTACAGCACAGGTCGATAAATCCAGCAAGTATATCT CGCACAG hTRA hTRA 139 ATGACATCCATTCGAGCTGTATTTATATTCCTGTGGCTGCAGCTGGAC TCCCT CCTGAAG V13-1 V13-1 TTGGTGAATGGAGAGAATGTGGAGCAGCATCCTTCAACCCTGAGTGTC GCACA ACTCGGC CAGGAGGGAGACAGCGCTGTTATCAAGTGTACTTATTCAGACAGTGCC TCACA TGTCTAC TCAAACTACTTCCCTTGGTATAAGCAAGAACTTGGAAAAGGACCTCAG GAGAC TTCTGTG CTTATTATAGACATTCGTTCAAATGTGGGCGAAAAGAAAGACCAACGA CCAA CAGCAAG ATTGCTGTTACATTGAACAAGACAGCCAAACATTTC TA hTRA hTRA 140 ATGGCAGGCATTCGAGCTTTATTTATGTACTTGTGGCTGCAGCTGGAC TCTCT CCTGGAG V13-2 V13-2 TGGGTGAGCAGAGGAGAGAGTGTGGGGCTGCATCTTCCTACCCTGAGT GCAAA ACTCAGC GTCCAGGAGGGTGACAACTCTATTATCAACTGTGCTTATTCAAACAGC TTGCA TGTCTAC GCCTCAGACTACTTCATTTGGTACAAGCAAGAATCTGGAAAAGGTCCT GCTAC TTTTGTG CAATTCATTATAGACATTCGTTCAAATATGGACAAAAGGCAAGGCCAA TCAA CAGAGAA AGAGTCACCGTTTTATTGAATAAGACAGTGAAACATCTC TA hTRA hTRA 141 ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTA TTGTC GGGACTC V14 V14 GGACCTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATGTTC ATCTC AGCAATG GTGCAGGAAAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGT CGCTT TATTTCT GATCAAAGTTATGGTCTATTCTGGTACAAGCAGCCCAGCAGTGGGGAA CACAA GTGCAAT ATGATTTTTCTTATTTATCAGGGGTCTTATGACGAGCAAAATGCAACA CTGG GAGAGAG GAAGGTCGCTACTCATTGAATTTCCAGAAGGCAAGAAAATCCGCCAAC GG C hTRA hTRA 142 ATGTATACGTATGTAACAAACCTGCGCGTTGTGCACATGTACCCTAGA GTTTT CCTGGAG V15 V15 ACGGGTGAACAGCCTCCATATTCTGGAGTAGAGTCCTTCATTCATTCC GAATA ATTCAGG TGAGTATCCGGGAGGGAATGCACAACATTCTTAATTGCACTTATGAGG TGCTG CACCTAC AGAGAACGTTCTCTTAACTTCTACTGGTTCTGGCAGGGTCTGGAAAAG GTCTC TTCTGTG GACTTGTGTCTTTGACCTTAATTCAATCAAGCCAGATGGAGGAGGGAG TCAT CTTTGAG ACAAACATTTTAAAGAAGCGCTTGGAAAAGAGAAGTTTTATAGT G hTRA hTRA 143 ATGAAGCCCACCCTCATCTCAGTGCTTGTGATAATATTTATACTCAGA CCTGA GGAAGAC V16 V16 GGAACAAGAGCCCAGAGAGTGACTCAGCCCGAGAAGCTCCTCTCTGTC AGAAA TCAGCCA TTTAAAGGGGCCCCAGTGGAGCTGAAGTGCAACTATTCCTATTCTGGG CCATT TGTATTA AGTCCTGAACTCTTCTGGTATGTCCAGTACTCCAGACAACGCCTCCAG TGCTC CTGTGCT TTACTCTTGAGACACATCTCTAGAGAGAGCATCAAAGGCTTCACTGCT AAGA CTAAGTG GACCTTAACAAAGGCGAGACATCTTTCCA G hTRA hTRA 144 ATGGAAACTCTCCTGGGAGTGTCTTTGGTGATTCTATGGCTTCAACTG TCCTT GCAGCAG V17 V17 GCTAGGGTGAACAGTCAACAGGGAGAAGAGGATCCTCAGGCCTTGAGC GTTGA ACACTGC ATCCAGGAGGGTGAAAATGCCACCATGAACTGCAGTTACAAAACTAGT TCACG TTCTTAC ATAAACAATTTACAGTGGTATAGACAAAATTCAGGTAGAGGCCTTGTC GCTTC TTCTGTG CACCTAATTTTAATACGTTCAAATGAAAGAGAGAAACACAGTGGAAGA CCGG CTACGGA TTAAGAGTCACGCTTGACACTTCCAAGAAAAGCAGT CG hTRA hTRA 145 ATGCTGTCTGCTTCCTGCTCAGGACTTGTGATCTTGTTGATATTCAGA ACCTG GCTGTCG V18 V18 AGGACCAGTGGAGACTCGGTTACCCAGACAGAAGGCCCAGTTACCCTC GAGAA GACTCTG CCTGAGAGGGCAGCTCTGACATTAAACTGCACTTATCAGTCCAGCTAT GCCCT CCGTGTA TCAACTTTTCTATTCTGGTATGTCCAGTATCTAAACAAAGAGCCTGAG CGGTG CTACTGC CTCCTCCTGAAAAGTTCAGAAAACCAGGAGACGGACAGCAGAGGTTTT CA GCTCTGA CAGGCCAGTCCTATCAAGAGTGACAGTTCCTTCC GA hTRA hTRA 146 ATGCTGACTGCCAGCCTGTTGAGGGCAGTCATAGCCTCCATCTGTGTT CACCA GGACTCA V19 V19 GTATCCAGCATGGCTCAGAAGGTAACTCAAGCGCAGACTGAAATTTCT TCACA GCAGTAT GTGGTGGAGAAGGAGGATGTGACCTTGGACTGTGTGTATGAAACCCGT GCCTC ACTTCTG GATACTACTTATTACTTATTCTGGTACAAGCAACCACCAAGTGGAGAA ACAAG TGCTCTG TTGGTTTTCCTTATTCGTCGGAACTCTTTTGATGAGCAAAATGAAATA TCGT AGTGAGG AGTGGTCGGTATTCTTGGAACTTCCAGAAATCCACCAGTTCCTTCAAC C TT hTRA hTRA 147 ATGGAGAAAATGTTGGAGTGTGCATTCATAGTCTTGTGGCTTCAGCTT TTTCT AACCTGA V20 V20 GGCTGGTTGAGTGGAGAAGACCAGGTGACGCAGAGTCCCGAGGCCCTG GCACA AGACTCA AGACTCCAGGAGGGAGAGAGTAGCAGTCTTAACTGCAGTTACACAGTC TCACA GCCACTT AGCGGTTTAAGAGGGCTGTTCTGGTATAGGCAAGATCCTGGGAAAGGC GCCCC ATCTCTG CCTGAATTCCTCTTCACCCTGTATTCAGCTGGGGAAGAAAAGGAGAAA TA TGCTGTG GAAAGGCTAAAAGCCACATTAACAAAGAAGGAAAGC CAGG hTRA hTRA 148 ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGG CTTTA TGGTGAC V21 V21 GTGAGCAGCAAACAGGAGGTGACGCAGATTCCTGCAGCTCTGAGTGTC TACAT TCAGCCA CCAGAAGGAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCT TGCAG CCTACCT ATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTCACA CTTCT CTGTGCT TCTCTGTTGCTTATTCAGTCAAGTCAGAGAGAGCAAACAAGTGGAAGA CAGCC GTGAGG CTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTA hTRA hTRA 149 ATGAAGAGGATATTGGGAGCTCTGCTGGGGCTCTTGAGTGCCCAGGTT GTACA AGACTCA V22 V22 TGCTGTGTGAGAGGAATACAAGTGGAGCAGAGTCCTCCAGACCTGATT TTTCC GGCGTTT CTCCAGGAGGGAGCCAATTCCACGCTGCGGTGCAATTTTTCTGACTCT TCTTC ATTTCTG GTGAACAATTTGCAGTGGTTTCATCAAAACCCTTGGGGACAGCTCATC AACCTGTTTTACATTCCCTCAGGGACAAAACAGAATGGAAGATTAAGC CCAGA TGCTGTG GCCACGACTGTCGCTACGGAACGCTACAGCTTATT CCAC GAGC hTRA hTRA 150 ATGGACAAGATCTTAGGAGCATCATTTTTAGTTCTGTGGCTTCAACTA CATTG CTGGAGA V23 V23 TGCTGGGTGAGTGGCCAACAGAAGGAGAAAAGTGACCAGCAGCAGGTG CATAT CTCAGCC AAACAAAGTCCTCAATCTTTGATAGTCCAGAAAGGAGGGATTTCAATT CATGG ACCTACT ATAAACTGTGCTTATGAGAACACTGCGTTTGACTACTTTCCATGGTAC ATTCC TCTGTGC CAACAATTCCCTGGGAAAGGCCCTGCATTATTGATAGCCATACGTCCA CAGC AGCAAGC GATGTGAGTGAAAAGAAAGAAGGAAGATTCACAATCTCCTTCAATAAA A AGTGCCAAGCAGTTCT hTRA hTRA 151 ATGGAGAAGAATCCTTTGGCAGCCCCATTACTAATCCTCTGGTTTCAT GCTAT AGCCTGA V24 V24 CTTGACTGCGTGAGCAGCATACTGAACGTGGAACAAAGTCCTCAGTCA TTGTA AGACTCA CTGCATGTTCAGGAGGGAGACAGCACCAATTTCACCTGCAGCTTCCCT CATCA GCCACAT TCCAGCAATTTTTATGCCTTACACTGGTACAGATGGGAAACTGCAAAA AAGGA ACCTCTG AGCCCCGAGGCCTTGTTTGTAATGACTTTAAATGGGGATGAAAAGAAG TCCC TGCCTTT AAAGGACGAATAAGTGCCACTCTTAATACCAAGGAGGGTTACA A hTRA hTRA 152 ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAG CAGCT CCCAGAC V25 V25 GTGAATGGACAACAGGTAATGCAAATTCCTCAGTACCAGCATGTACAA CCCTG TACAGAT GAAGGAGAGGACTTCACCACGTACTGCAATTCCTCAACTACTTTAAGC CACAT GTAGGAA AATATACAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTG CACAG CCTACTT ATACAGTTAGTGAAGAGTGGAGAAGTGAAGAAGCAGAAAAGACTGACA CCA CTGTGCA TTTCAGTTTGGAGAAGCAAAAAAGAA GGG hTRA hTRA 153 ATGAGGCTGGTGGCAAGAGTAACTGTGTTTCTGACCTTTGGAACTATA TTGAT GAGACAC V26-1 V26-1 ATTGATGCTAAGACCACCCAGCCCCCCTCCATGGATTGCGCTGAAGGA CCTGC TGCTGTG AGAGCTGCAAACCTGCCTTGTAATCACTCTACCATCAGTGGAAATGAG CCCAC TACTATT TATGTGTATTGGTATCGACAGATTCACTCCCAGGGGCCACAGTATATC GCTAC GCATCGT ATTCATGGTCTAAAAAACAATGAAACCAATGAAATGGCCTCTCTGATC GCTGA CAGAGTC ATCACAGAAGACAGAAAGTCCAGCACC G hTRA hTRA 154 ATGAAGTTGGTGACAAGCATTACTGTACTCCTATCTTTGGGTATTATG TTGAT GAGATGC V26-2 V26-2 GGTGATGCTAAGACCACACAGCCAAATTCAATGGAGAGTAACGAAGAA CCTGC TGCTGTG GAGCCTGTTCACTTGCCTTGTAACCACTCCACAATCAGTGGAACTGAT ACCGT TACTACT TACATACATTGGTATCGACAGCTTCCCTCCCAGGGTCCAGAGTACGTG GCTAC GCATCCT ATTCATGGTCTTACAAGCAATGTGAACAACAGAATGGCCTCTCTGGCA CTTGA GAGAGAC ATCGCTGAAGACAGAAAGTCCAGTACC hTRA hTRA 155 ATGGTCCTGAAATTCTCCGTGTCCATTCTTTGGATTCAGTTGGCATGG GTTCT CAGCCTG V27 V27 GTGAGCACCCAGCTGCTGGAGCAGAGCCCTCAGTTTCTAAGCATCCAA CTCCA GTGATAC GAGGGAGAAAATCTCACTGTGTACTGCAACTCCTCAAGTGTTTTTTCC CATCA AGGCCTC AGCTTACAATGGTACAGACAGGAGCCTGGGGAAGGTCCTGTCCTCCTG CTGCA TACCTCT GTGACAGTAGTTACGGGTGGAGAAGTGAAGAAGCTGAAGAGACTAACC GCC GTGCAGG TTTCAGTTTGGTGATGCAAGAAAGGACA AG hTRA hTRA 156 ATGAAGGCATTAATAGGAATCTTGCTGGGCTTCCTGTGGATACAGATT GCCAC GCCTGAG V28 V28 TGCTCGCAAATGAAAGTGGAGCAGAGTCCTCAGGTCCTGATCCTCCAA CTATA GACTCAG GAGGGAAGAAATTCATTCCTGGTGTGCAGTTGTTCTATTTACATGATC CATCA CTATTTA CGTGTGCAGTGGTTTCATCAAAAGCCTGGAGGACCCCTCATGTCCTTA GATTC CTTCTGT TTTAACATTAATTCAGGAATACAGCAAAAAAGAAGACTAAAATCCGCA CCA GCTGTGG GTCAAAGCTGAGGAACTTTATG GGA hTRA hTRA 157 ATGGCCATGCTCCTGGGGGCATCAGTGCTGATTCTGTGGCTTCAGCCA TCTCT GCCTGGA V29 V29 GACTGGGTAAACAGTCAACAGAAGAATGATGACCAGCAAGTTAAGCAA GCACA GACTCTG AATTCACCATCCCTGAGCGTCCAGGAAGGAAGAATTTCTATTCTGAAC TTGTG CAGTGTA TGTGACTATACTAACAGCATGTTTGATTATTTCCTATGGTACAAAAAA CCCTC CTTCTGT TACCCTGCTGAAGGTCCTACATTCCTGATATCTATAAGTTCCATTAAG CCA GCAGCAA GATAAAAATGAAGATGGAAGATTCACTGTCTTCTTAAACAAAAGTGCC GCG AAGCACCTC hTRA hTRA 158 ATGGAGACTCTCCTGAAAGTGCTTTCAGGCACCTTGTTGTGGCAGTTG CCCTG CAGTTAC V30 V30 ACCTGGGTGAGAAGCCAACAACCAGTGCAGAGTCCTCAAGCCGTGATC TACCT TCAGGAA CTCCGAGAAGGGGAAGATGCTGTCATCAACTGCAGTTCCTCCAAGGCT TACGG CCTACTT TTATATTCTGTACACTGGTACAGGCAGAAGCATGGTGAAGCACCCGTC CCTCC CTGCGGC TTCCTGATGATATTACTGAAGGGTGGAGAACAGAAGGGTCATGAAAAA CAGCT ACAGAGA ATATCTGCTTCATTTAATGAAAAAAAGCAGCAAAGCT hTRA hTRA 159 ATGACTGTTGGCAGCATATTACGGGCACTCATGGCCTCTGCCTTCCTT CTTAT GAAGACC V31 V31 GCATGTCACAGAGGGTCATTCAATCCCAACCAGCAATATCTACGCAGG CATAT TGCAACA AGGGTGAGACCGTGAAACTGGACTGTGCATACAAAACTAATATTGTAT CATCA TATTTCT ATTACATATTGTATTGGTACAAAAGGTCTCCCAATGGGAAGATTATTT TCACA GTTGTCT TCCTCATTTATCAGCAAACAGATGCAGAAACCAATGCGACACAGGGTC GCCA CAAAGAG AATATTCTGTGAGCTTCCAGAAAACAACTAAAACTATTCAG CC hTRA hTRA 160 ATGGCAAGAAGAATGGAAAAGTCCCTGGGAGCTTTATTCAAATTCAGC TCCCT CCAGGAG V32 V32 TGAAGCTGGCCAAGAAAAGGATGTGATACAGAGTTATTCAAATCTAAA GCATA ACTCATT TGTCTAGGAGAGAGAAATGGCCGTTATTAATGACAGTTATACAGATGG TTACA CCTGTAC AGCTTTGAATTATTTCTGTTGGTACAAGAAGAAAACGGGGAAGGCCCT GCCAC TTCTGTG AATATCTTAATGGAGATTCATTCAAATGTGGATAGAAAACAGGACAGA CCAA CAGTGAG AGGCTCACTGTACTGTTGAATAAAAATGCTAAACATGTC AACACA hTRA hTRA 161 ATGCTCTGCCCTGGCCTGCTGTGGGCATTCGTGGTCCCCTTTGGCTTC ACCTC TGACTCA V33 V33 AGATCCAGCATGGCTCAGAAAGTAACCCAAGTTCAGACCACAGTAACT ACCAT GCCAAGT AGGCAGAAAGGAGTAGCTGTGACCTTGGACTGCATGTTTGAAACCAGA CAATT ACTTCTG TAGAATTCGTACACTTTATACTGGTACAAGCAACAAGCAACCTCCCAG CCTTA TGCTCTC TGAAGAGATGGTTTTCCTTATTCATCAGGGTTATTCTAAGTCAAATGC AAAC AGGAATC AAAGCCTGTGAACTTTGAAAAAAAGAAAAAGTTCATCA C hTRA hTRA 162 ATGGAGACTGTTCTGCAAGTACTCCTAGGGATATTGGGGTTCCAAGCA TCCCT CCCAGCC V34 V34 GCCTGGGTCAGTAGCCAAGAACTGGAGCAGAGTCCTCAGTCCTTGATC GCATA ATGCAGG GTCCAAGAGGGAAAGAATCTCACCATAAACTGCACGTCATCAAAGACG TCACA CATCTAC TTATATGGCTTATACTGGTATAAGCAAAAGTATGGTGAAGGTCTTATC GCCTC CTCTGTG TTCTTGATGATGCTACAGAAAGGTGGGGAAGAGAAAAGTCATGAAAAG CCAG GAGCAGA ATAACTGCCAAGTTGGATGAGAAAAAGCAGCAAAGT CA hTRA hTRA 163 ATGCTCCTTGAACATTTATTAATAATCTTGTGGATGCAGCTGACATGG CTTCC ACCTAGT V35 V35 GTCAGTGGTCAACAGCTGAATCAGAGTCCTCAATCTATGTTTATCCAG TGAAT GATGTAG GAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAAC ATCTC GCATCTA ACCTGGCTATGGTACAAGCAGGAACCTGGGGAAGGTCCTGTCCTCTTG AGCAT CTTCTGT ATAGCCTTATATAAGGCTGGTGAATTGACCTCAAATGGAAGACTGACT CCAT GCTGGGC GCTCAGTTTGGTATAACCAGAAAGGACAG AG hTRA hTRA 164 ATGATGAAGTGTCCACAGGCTTTACTAGCTATCTTTTGGCTTCTACTG TCCTG ACCGGAG V36 V36 AGCTGGGTGAGCAGTGAAGACAAGGTGGTACAAAGCCCTCTATCTCTG AACAT ACTCGGC GTTGTCCACGAGGGAGACACCGTAACTCTCAATTGCAGTTATGAAGTG CACAG CATCTAC ACTAACTTTCGAAGCCTACTATGGTACAAGCAGGAAAAGAAAGCTCCC CCACC CTCTGTG ACATTTCTATTTATGCTAACTTCAAGTGGAATTGAAAAGAAGTCAGGA CAG CTGTGGA AGACTAAGTAGCATATTAGATAAGAAAGAACTTTCCAGCA GG hTRA hTRA 165 ATGGAAACTCCACTGAGCACTCTGCTGCTGCTCCTCTGTGTGCAGCTG TCCCT CTCCATG V37 V37 ACCTGGTCAAATGGACAACTGCCAGTGGAACAGAATGCTCCTTCCCTG GCACA ACTCAAC AAAGTCAAGGAAGGTGACAGCGTCACACTGAACTGCAGTTACAGAGAC TACAG CACATTC AGCCCTTCAGATTTCTTCAGTGGTTCAGGCAGGATCCTGAGGAAGGCC GATTC TTCTGCG TCATTTCCCTGATACAAATGCTATCAACTGTGAGAGAGAAGATCAGTG CCAG CAGCAAG GAAGATTCACAGCCAGGCTTAAAAAAGGAGACCAGCACATT CA hTRA hTRA 166 ATGACACGAGTTAGCTTGCTGTGGGCAGTCGTGGTCTCCACCTGTCTT CAAGA GGGACAC V38-1 V38 GAATCCGGCATGGCCCAGACAGTCACTCAGTCTCAACCAGAGATGTCT TCTCA TGCGATG GTGCAGGAGGCAGAGACTGTGACCCTGAGTTGCACATATGACACCAGT GACTC TATTTCT GAGAATAATTATTATTTGTTCTGGTACAAGCAGCCTCCCAGCAGGCAG ACAGC GTGCTTT ATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACG TGG CATGAAG GAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGT CA CT hTRA hTRA 167 ATGGCATGCCCTGGCTTCCTGTGGGCACTTGTGATCTCCACCTGTCTT CAAGA GGGATGC V38-2 V38 GAATTTAGCATGGCTCAGACAGTCACTCAGTCTCAACCAGAGATGTCT TCTCA CGCGATG GTGCAGGAGGCAGAGACCGTGACCCTGAGCTGCACATATGACACCAGT GACTC TATTTCT GAGAGTGATTATTATTTATTCTGGTACAAGCAGCCTCCCAGCAGGCAG ACAGC GTGCTTA ATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACA TGG TAGGAGC GAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGT G CT hTRA hTRA 168 ATGAAGAAGCTACTAGCAATGATTCTGTGGCTTCAACTAGACCGGTTA CCGTC CAGCTGC V39 V39 AGTGGAGAGCTGAAAGTGGAACAAAACCCTCTGTTCCTGAGCATGCAG TCAGC CGTGCAT GAGGGAAAAAACTATACCATCTACTGCAATTATTCAACCACTTCAGAC ACCCT GACCTCT AGACTGTATTGGTACAGGCAGGATCCTGGGAAAAGTCTGGAATCTCTG CCACA CTGCCAC TTTGTGTTGCTATCAAATGGAGCAGTGAAGCAGGAGGGACGATTAATG TCA CTACTTC GCCTCACTTGATACCAAAGC TGTGCCG TGGACA hTRA hTRA 169 ATGAACTCCTCTCTGGACTTTCTAATTCTGATCTTAATGTTTGGAGGA CCATT TATCAGA V40 V40 ACCAGCAGCAATTCAGTCAAGCAGACGGGCCAAATAACCGTCTCGGAG GTGAA CTCAGCC GGAGCATCTGTGACTATGAACTGCACATACACATCCACGGGGTACCCT ATATT GTGTACT ACCCTTTTCTGGTATGTGGAATACCCCAGCAAACCTCTGCAGCTTCTT CAGTC ACTGTCT CAGAGAGAGACAATGGAAAACAGCAAAAACTTCGGAGGCGGAAATATT CAGG TCTGGGA AAAGACAAAAACTCCC GA hTRA hTRA 170 ATGGTGAAGATCCGGCAATTTTTGTTGGCTATTTTGTGGCTTCAGCTA CTGCA TCCCAGA V41 V10 AGCTGTGTAAGTGCCGCCAAAAATGAAGTGGAGCAGAGTCCTCAGAAC CATCA GACTCTG CTGACTGCCCAGGAAGGAGAATTTATCACAATCAACTGCAGTTACTCG CAGCC CCGTCTA GTAGGAATAAGTGCCTTACACTGGCTGCAACAGCATCCAGGAGGAGGC TCCCA CATCTGT ATTGTTTCCTTGTTTATGCTGAGCTCAGGGAAGAAGAAGCATGGAAGA GCTGTCA TTAATTGCCACAATAAACATACAGGAAAAGCACAGCTCC GA

TABLE 16 TCR β chain V segments and binding sites for primers presented in Table 4 and Table 10. The sequence for each V segments presented in this table consists of three parts (listed from 5′ to 3′): Sequence upstream of primer binding site, sequence of the primer binding site and sequence downstream of the primer binding site. hTRBV Primer sequence binding downstream V site of primer segment Primer SEQ within binding name name ID NO hTRBV sequence upstream of primer binding site hTRBV site hTRB hTRB 171 ATGGGCTGAAGTCTCCACTGTGGTGTGGTCCATTGTCTCAGGCTCCAT GTGGT CTCAGCT V01 V01 GGATACTGGAATTACCCAGACACCAAAATACCTGGTCACAGCAATGGG CGCAC GCGTATC GAGTAAAAGGACAATGAAACGTGAGCATCTGGGACATGATTCTATGTA TGCAG TCTGCAC TTGGTACAGACAGAAAGCTAAGAAATCCCTGGAGTTCATGTTTTACTA CAAGA CAGCAGC CAACTGTAAGGAATTCATTGAAAACAAGACTGTGCCAAATCACTTCAC AGA CAAGA ACCTGAATGCCCTGACAGCTCTCGCTTATACCTTCAT hTRB hTRB 172 ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCA GATCC CTCAGCC V02 V02 GGACTCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTCACA GGTCC ATGTACT CAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCAC ACAAA TCTGTGC TTATACTTCTATTGGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTT GCTGG CAGCAGT CTGGTTTCCTTTTATAATAATGAAATCTCAGAGAAGTCTGAAATATTC AGGA GAAGC GATGATCAATTCTCAGTTGAAAGGCCTGATGGATCAAATTTCACTCTG AA hTRB hTRB 173 ATGGGCTGCAGGCTCCTCTGCTGTGTGGTCTTCTGCCTCCTCCAAGCA CATCA CTCTGCT V03-1 V03-1 GGTCCCTTGGACACAGCTGTTTCCCAGACTCCAAAATACCTGGTCACA ATTCC GTGTATT CAGATGGGAAACGACAAGTCCATTAAATGTGAACAAAATCTGGGCCAT CTGGA TCTGTGC GATACTATGTATTGGTATAAACAGGACTCTAAGAAATTTCTGAAGATA GCTTG CAGCAGC ATGTTTAGCTACAATAATAAGGAGCTCATTATAAATGAAACAGTTCCA GTGA CAAGA AATCGCTTCTCACCTAAATCTCCAGACAAAGCTCACTTAAATCTTCA hTRB hTRB 174 ATGGGCTGCAGGCTCCTCTGCTATGTGGCCCTCTGCCTCCTGCAAGCA CATCA CTCTGCT V03-2 V03-1 GGATCCACTGGACACAGCCGTTTCCCAGACTCCAAAATACCTGGTCAC ATTCC GTGTATT ACAGATGGGAAAAAAGGAGTCTCTTAAATGAGAACAAAATCTGGGCCA CTGGA TCTGTGC TAATGCTATGTATTGGTATAAACAGGACTCTAAGAAATTTCTGAAGAC GCTTG CAGCAGC AATGTTTATCTACAGTAACAAGGAGCCAATTTTAAATGAAACAGTTCC GTGA CAAGA AAATCGCTTCTCACCTGACTCTCCAGACAAAGCTCATTTAAATCTTCA hTRB hTRB 175 ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCA TTCAC AAGACTC V04-1 V04-1 GTTCCCATAGACACTGAAGTTACCCAGACACCAAAACACCTGGTCATG CTACA AGCCCTG GGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATATGGGGCAC CGCCC TATCTCT AGGGCTATGTATTGGTACAAGCAGAAAGCTAAGAAGCCACCGGAGCTC TGCAG GCGCCAG ATGTTTGTCTACAGCTATGAGAAACTCTCTATAAATGAAAGTGTGCCA CCAG CAGCCAA AGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCTCTTAAACC GA hTRB hTRB 176 ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCG TTCAC AAGACTC V04-2 V04-2 GTCCCCATGGAAACGGGAGTTACGCAGACACCAAGACACCTGGTCATG CTACA GGCCCTG GGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATCTGGGGCAT CACCC TATCTCT AACGCTATGTATTGGTACAAGCAAAGTGCTAAGAAGCCACTGGAGCTC TGCAG GTGCCAG ATGTTTGTCTACAACTTTAAAGAACAGACTGAAAACAACAGTGTGCCA CCAG CAGCCAA AGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCACTTATTCC GA hTRB hTRB 177 ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCG TTCAC AAGACTC V04-3 V04-2 GGTGAGTTGGTCCCCATGGAAACGGGAGTTACGCAGACACCAAGACAC CTACA GGCCCTG CTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACAT CACCC TATCTCT CTGGGTCATAACGCTATGTATTGGTACAAGCAAAGTGCTAAGAAGCCA TGCAG GCGCCAG CTGGAGCTCATGTTTGTCTACAGTCTTGAAGAACGGGTTGAAAACAAC CCAG CAGCCAA AGTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCACTTA GA TTCC hTRB hTRB 178 ATGGGCTCCAGGCTGCTCTGTTGGGTGCTGCTTTGTCTCCTGGGAGCA GAATG GGGACTC V05-1 V05-1 GGCCCAGTAAAGGCTGGAGTCACTCAAACTCCAAGATATCTGATCAAA TGAGC GGCCCTT ACGAGAGGACAGCAAGTGACACTGAGCTGCTCCCCTATCTCTGGGCAT ACCTT TATCTTT AGGAGTGTATCCTGGTACCAACAGACCCCAGGACAGGGCCTTCAGTTC GGAGC GCGCCAG CTCTTTGAATACTTCAGTGAGACACAGAGAAACAAAGGAAACTTCCCT TGG CAGCTTG GGTCGATTCTCAGGGCGCCAGTTCTCTAACTCTCGCTCTGAGAT G hTRB hTRB 179 ATGGGCTCCGGACTCCTCTGCTGGACGCTGCTTTGTTTCCTGGGAGCA TACTG GGACTCA V05-2 V05-2 GGCCCAGTGGAGGCTGGAATCACCCAAGCTCCAAGACACCTGATCAAA AGTCA GCCCTGT ACAAGAGACCAGCAAGTGACACTGAGATGCTCCCCTGCCTCTGGGCAT AACAC ATCTCTG AACTGTGTGTCCTGGTACCTACGAACTCCAAGTCAGCCCCTCTAGTTA TTGTTACAATATTGTAATAGGTTACAAAGAGCAAAAGGAAACTTGCCT GGAGC TGCCAGC AATTGATTCTCAGCTCACCACGTCCATAACTAT TAGG AACTTG hTRB hTRB 180 ATGGGCCCCGGGCTCCTCTGCTGGGAACTGCTTTATCTCCTGGGAGCA GCTCT TGGAGCT V05-3 V05-3 GGCCCAGTGGAGGCTGGAGTCACCCAAAGTCCCACACACCTGATCAAA GAGAT GGGGGAC ACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTATCTCTGGGCAC GAATG TCGGCCC AGCAGTGTGTCCTGGTACCAACAGGCCCCGGGTCAGGGGCCCCAGTTT TGAGT TGTATCT ATCTTTGAATATGCTAATGAGTTAAGGAGATCAGAAGGAAACTTCCCT GCCT CTGTGCC AATCGATTCTCAGGGCGCCAGTTCCATGACTGTT AGAAGCT TGG hTRB hTRB 181 ATGGGCCCTGGGCTCCTCTGCTGGGTGCTGCTTTGTCTCCTGGGAGCA CTGAG GGAGCTG V05-4 V05-4 GGCTCAGTGGAGACTGGAGTCACCCAAAGTCCCACACACCTGATCAAA CTGAA GACGACT ACGAGAGGACAGCAAGTGACTCTGAGATGCTCTTCTCAGTCTGGGCAC TGTGA CGGCCCT AACACTGTGTCCTGGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTT ACGCC GTATCTC ATCTTTCAGTATTATAGGGAGGAAGAGAATGGCAGAGGAAACTTCCCT TT TGTGCCA CCTAGATTCTCAGGTCTCCAGTTCCCTAATTATAGCT GCAGCTT GG hTRB hTRB 182 ATGGGCCCTGGGCTCCTCTGCTGGGTGCTGCTTTGTCTCCTGGGAGCA CTGAG GTTGCTG V05-5 V05-4 GGCCCAGTGGACGCTGGAGTCACCCAAAGTCCCACACACCTGATCAAA CTGAA GGGGACT ACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTATCTCTGGGCAC TGTGA CGGCCCT AAGAGTGTGTCCTGGTACCAACAGGTCCTGGGTCAGGGGCCCCAGTTT ACGCC GTATCTC ATCTTTCAGTATTATGAGAAAGAAGAGAGAGGAAGAGGAAACTTCCCT TT TGTGCCA GATCGATTCTCAGCTCGCCAGTTCCCTAACTATAGCT GCAGCTT GG hTRB hTRB 183 ATGGGCCCCGGGCTCCTCTGCTGGGCACTGCTTTGTCTCCTGGGAGCA CTGAG GTTGCTG V05-6 V05-4 GGCTTAGTGGACGCTGGAGTCACCCAAAGTCCCACACACCTGATCAAA CTGAA GGGGACT ACGAGAGGACAGCAAGTGACTCTGAGATGCTCTCCTAAGTCTGGGCAT TGTGA CGGCCCT GACACTGTGTCCTGGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTT ACGCC CTATCTC ATCTTTCAGTATTATGAGGAGGAAGAGAGACAGAGAGGCAACTTCCCT TT TGTGCCA GATCGATTCTCAGGTCACCAGTTCCCTAACTATAGCT GCAGCTT GG hTRB hTRB 184 ATGGGCCCCGGGCTCCTCTGCTGGGTGCTGCTTTGTCCCCTAGGAGAA CTGAG GTTGCTA V05-7 V05-4 GGCCCAGTGGACGCTGGAGTCACCCAAAGTCCCACACACCTGATCAAA CTGAA GGGGACT ACGAGAGGACAGCACGTGACTCTGAGATGCTCTCCTATCTCTGGGCAC TGTGA CGGCCCT ACCAGTGTGTCCTCGTACCAACAGGCCCTGGGTCAGGGGCCCCAGTTT ACGCC CTATCTC ATCTTTCAGTATTATGAGAAAGAAGAGAGAGGAAGAGGAAACTTCCCT TT TGTGCCA GATCAATTCTCAGGTCACCAGTTCCCTAACTATAGCT GCAGCTT GG hTRB hTRB 185 ATGGGACCCAGGCTCCTCTTCTGGGCACTGCTTTGTCTCCTCGGAACA CTGAG GGAGCTG V05-8 V05-4 GGCCCAGTGGAGGCTGGAGTCACACAAAGTCCCACACACCTGATCAAA CTGAA GAGGACT ACGAGAGGACAGCAAGCGACTCTGAGATGCTCTCCTATCTCTGGGCAC TGTGA CGGCCCT ACCAGTGTGTACTGGTACCAACAGGCCCTGGGTCTGGGCCTCCAGTTC ACGCC GTATCTC CTCCTTTGGTATGACGAGGGTGAAGAGAGAAACAGAGGAAACTTCCCT TT TGTGCCA CCTAGATTTTCAGGTCGCCAGTTCCCTAATTATAGCT GCAGCTT GG hTRB hTRB 186 ATGAGCATCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCA GAGTT CGGCTGC V06-1 V06-1 AGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTG CTCGC TCCCTCC AAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCAT TCAGG CAGACAT AACTCCATGTACTGGTATCGACAAGACCCAGGCATGGGACTGAGGCTG CTGGA CTGTGTA ATTTATTACTCAGCTTCTGAGGGTACCACTGACAAAGGAGAAGTCCCC GT CTTCTGT AATGGCTACAATGTCTCCAGATTAAACAAACGG GCCAGCA GTGAAGC hTRB hTRB 187 ATGAGCCTCGGGCTCCTGTGCTGTGGGGCCTTTTCTCTCCTGTGGGCA CTGGG CCTCCCA V06-2 V06-2 GGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCGGGTCCTG GTTGG AACATCT AAGACAGGACAGAGCATGACACTGCTGTGTGCCCAGGATATGAACCAT AGTCG GTGTACT GAATACATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTG GCTGC TCTGTGC ATTCATTACTCAGTTGGTGAGGGTACAACTGCCAAAGGAGAGGTCCCT TC CAGCAGT GATGGCTACAATGTCTCCAGATTAAAAAAACAGAATTTCCTG TACTC hTRB hTRB 188 ATGAGCCTCGGGCTCCTGTGCTGTGGGGCCTTTTCTCTCCTGTGGGCA CTGGG CCTCCCA V06-3 V06-2 GGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCGGGTCCTG GTTGG AACATCT AAGACAGGACAGAGCATGACACTGCTGTGTGCCCAGGATATGAACCAT AGTCG GTGTACT GAATACATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTG GCTGC TCTGTGC ATTCATTACTCAGTTGGTGAGGGTACAACTGCCAAAGGAGAGGTCCCT TC CAGCAGT GATGGCTACAATGTCTCCAGATTAAAAAAACAGAATTTCCTG TACTC hTRB hTRB 189 ATGAGAATCAGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCA CCCCT TACCCTC V06-4 V06-4 GGTCCAGTGATTGCTGGGATCACCCAGGCACCAACATCTCAGATCCTG CACGT TCAGACA GCAGCAGGACGGCGCATGACACTGAGATGTACCCAGGATATGAGACAT TGGCG TCTGTGT AATGCCATGTACTGGTATAGACAAGATCTAGGACTGGGGCTAAGGCTC TCTGC ACTTCTG ATCCATTATTCAAATACTGCAGGTACCACTGGCAAAGGAGAAGTCCCT TG TGCCAGC GATGGTTATAGTGTCTCCAGAGCAAACACAGATGATTTC AGTGACT C hTRB hTRB 190 ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCA TCCCG TGCTCCC V06-5 V06-5 GGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTG CTCAG TCCCAGA AAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCAT GCTGC CATCTGT GAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTG TGTCG GTACTTC ATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCC GC TGTGCCA AATGGCTACAATGTCTCCAGATCAACCACAGAGGATT GCAGTTA CTC hTRB hTRB 191 ATGAGCATCAGCCTCCTGTGCTGTGCAGCCTTTCCTCTCCTGTGGGCA GATTT TGGCTGC V06-6 V06-6 GGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCGCATCCTG CCCGC TCCCTCC AAGATAGGACAGAGCATGACACTGCAGTGTACCCAGGATATGAACCAT TCAGG CAGACAT AACTACATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAAGCTG CTGGA CTGTGTA ATTTATTATTCAGTTGGTGCTGGTATCACTGATAAAGGAGAAGTCCCG GT CTTCTGT AATGGCTACAACGTCTCCAGATCAACCACAGAG GCCAGCA GTTACTC hTRB hTRB 192 ATGAGCCTCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCA TCCCC GCTCCCT V06-7 V06-7 GGTCCAATGAATGCTGGTGTCACTCAGACCCCAAAATTCCACGTCCTG CTCAA CTCAGAC AAGACAGGACAGAGCATGACTCTGCTGTGTGCCCAGGATATGAACCAT GCTGG TTCTGTTT GAATACATGTATCGGTATCGACAAGACCCAGGCAAGGGGCTGAGGCTG AGTCA ACTTCTG ATTTACTACTCAGTTGCTGCTGCTCTCACTGACAAAGGAGAAGTTCCC GCT TGCCAGC AATGGCTACAATGTCTCCAGATCAAACACAGAGGATT AGTTACT C hTRB hTRB 193 ATGAGCCTCGGGCTCCTGTGCTGTGCGGCCTTTTCTCTCCTGTGGGCA TCCCA TGCTCCC V06-8 V06-8 GGTCCCGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCACATCCTG CTCAG TCCCAGA AAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCAT GCTGG CATCTGT GGATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGACTG TGTCG GTACTTG ATTTACTACTCAGCTGCTGCTGGTACTACTGACAAAGAAGTCCCCAAT GC TGTGCCA GGCTACAATGTCTCTAGATTAAACACAGAGGATT GCAGTTA CTC hTRB hTRB 194 ATGAGCATCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGAG GATTT CAGCTGC V06-9 V06-6 GGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCACATCCTG CCCGC TCCCTCC AAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCAT TCAGG CAGACAT GGATACTTGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCGC CTGGA CTGTATA ATTCATTACTCAGTTGCTGCTGGTATCACTGACAAAGGAGAAGTCCCC GT CTTCTGT GATGGCTACAATGTATCCAGATCAAACACAGAG GCCAGCA GTTATTC hTRB hTRB 195 ATGGGCACAAGGCTCCTCTGCTGGGCAGCCATATGTCTCCTGGGGGCA CTCTG GCAGGGG V07-1 V07-1 GATCACACAGGTGCTGGAGTCTCCCAGTCCCTGAGACACAAGGTAGCA AAGTT GACTTGG AAGAAGGGAAAGGATGTAGCTCTCAGATATGATCCAATTTCAGGTCAT CCAGC CTGTGTA AATGCCCTTTATTGGTACCGACAGAGCCTGGGGCAGGGCCTGGAGTTT GCACA TCTCTGT CCAATTTACTTCCAAGGCAAGGATGCAGCAGACAAATCGGGGCTTCCC CA GCCAGCA CGTGATCGGTTCTCTGCACAGAGGTCTGAGGGATCCATCTCCA GCTCAGC hTRB hTRB 196 ATGGGCACCAGGCTCCTCTTCTGGGTGGCCTTCTGTCTCCTGGGGGCA GATCC GACTCGG V07-2 V07-2 GATCACACAGGAGCTGGAGTCTCCCAGTCCCCCAGTAACAAGGTCACA AGCGC CCGTGTA GAGAAGGGAAAGGATGTAGAGCTCAGGTGTGATCCAATTTCAGGTCAT ACACA TCTCTGT ACTGCCCTTTACTGGTACCGACAGAGCCTGGGGCAGGGCCTGGAGTTT GCAGG GCCAGCA TTAATTTACTTCCAAGGCAACAGTGCACCAGACAAATCAGGGCTGCCC AG GCTTAGC AGTGATCGCTTCTCTGCAGAGAGGACTGGGGGATCCGTCTCCACTCTG AC hTRB hTRB 197 ATGGGCACCAGGCTCCTCTGCTGGGCAGCCCTGTGCCTCCTGGGGGCA ACTCT GCGGGGG V07-3 V07-5 GATCACACAGGTGCTGGAGTCTCCCAGACCCCCAGTAACAAGGTCACA GAAGA GACTCAG GAGAAGGGAAAATATGTAGAGCTCAGGTGTGATCCAATTTCAGGTCAT TCCAG CCGTGTA ACTGCCCTTTACTGGTACCGACAAAGCCTGGGGCAGGGCCCAGAGTTT CGCAC TCTCTGT CTAATTTACTTCCAAGGCACGGGTGCGGCAGATGACTCAGGGCTGCCC AGA GCCAGCA AACGATCGGTTCTTTGCAGTCAGGCCTGAGGGATCCGTCTCT GCTTAAC hTRB hTRB 198 ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACA ACTCT GCAGGGG V07-4 V07-5 GATCACACAGGTGCTGGAGTCTCCCAGTCCCCAAGGTACAAAGTCGCA GAAGA GACTCAG AAGAGGGGACGGGATGTAGCTCTCAGGTGTGATTCAATTTCGGGTCAT TCCAG CTGTGTA GTAACCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCTCAGAGGTT CGCAC TCTCTGT CTGACTTACTCCCAGAGTGATGCTCAACGAGACAAATCAGGGCGGCCC AGA GCCAGCA AGTGGTCGGTTCTCTGCAGAGAGGCCTGAGAGATCCGTCTCC GCTTAGC hTRB hTRB 199 ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACA AGATC GCGACTC V07-5 V07-5 GATCACACAGGTGCTGGAGTCTCCCAGTCCCCAAGGTACGAAGTCACA CAGCG GGCTGTG CAGAGGGGACAGGATGTAGCTCCCAGGTGTGATCCAATTTCGGGTCAG CACAG TATCTCT GTAACCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCAAGAGTTT AGCAA GTGCCAG CTGACTTCCTTCCAGGATGAAACTCAACAAGATAAATCAGGGCTGCTC GG AAGCTTA AGTGATCAATTCTCCACAGAGAGGTCTGAGGATCTTTCTCCACCTGA G hTRB hTRB 200 ATGGGCACCAGTCTCCTATGCTGGGTGGTCCTGGGTTTCCTAGGGACA CAGCG CGGCCAT V07-6 V07-6 GATCACACAGGTGCTGGAGTCTCCCAGTCTCCCAGGTACAAAGTCACA CACAG GTATCGC AAGAGGGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCAT AGCAG TGTGCCA GTATCCCTTTATTGGTACCGACAGGCCCTGGGGCAGGGCCCAGAGTTT CGGGA GCAGCTT CTGACTTACTTCAATTATGAAGCCCAACAAGACAAATCAGGGCTGCCC CT AGC AATGATCGGTTCTCTGCAGAGAGGCCTGAGGGATCCATCTCCACTCTG ACGATC hTRB hTRB 201 ATGGGTACCAGTCTCCTATGCTGGGTGGTCCTGGGTTTCCTAGGGACA CAGCG CAGCCAT V07-7 V07-6 GATCACACAGGTGCTGGAGTCTCCCAGTCTCCCAGGTACAAAGTCACA CACAG GTATCGC AAGAGGGGACAGGATGTAACTCTCAGGTGTGATCCAATTTCGAGTCAT AGCAG TGTGCCA GCAACCCTTTATTGGTATCAACAGGCCCTGGGGCAGGGCCCAGAGTTT CGGGA GCAGCTT CTGACTTACTTCAATTATGAAGCTCAACCAGACAAATCAGGGCTGCCC CT AGC AGTGATCGGTTCTCTGCAGAGAGGCCTGAGGGATCCATCTCCACTCTG ACGATT hTRB hTRB 202 ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACA GATCC GACTCCG V07-8 V07-2 GATCACACAGGTGCTGGAGTCTCCCAGTCCCCTAGGTACAAAGTCGCA AGCGC CCGTGTA AAGAGAGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCAT ACACA TCTCTGT GTATCCCTTTTTTGGTACCAACAGGCCCTGGGGCAGGGGCCAGAGTTT GCAGG GCCAGCA CTGACTTATTTCCAGAATGAAGCTCAACTAGACAAATCGGGGCTGCCC AG GCTTAGC AGTGATCGCTTCTTTGCAGAAAGGCCTGAGGGATCCGTCTCCACTCTG AA hTRB hTRB 203 ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCA GAGAT GGGACTC V07-9 V07-9 GATCACGCAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACA CCAGC GGCCATG AAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACAC GCACA TATCTCT AACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTT GAGCA GTGCCAG CTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTC GG CAGCTTA AGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTG GC hTRB hTRB 204 GAGGCAGGGATCAGCCAGATACCAAGATATCACAGACACACAGGGAAA CCCTC GCACCAG V08-1 V08-1 AAGATCATCCTGAAATATGCTCAGATTAGGAACCATTATTCAGTGTTC AACCC CCAGACC TGTTATCAATAAGACCAAGAATAGGGGCTGAGGCTGATCCATTATTCA TGGAG TCTGTAC GGTAGTATTGGCAGCATGACCAAAGGCGGTGCCAAGGAAGGGTACAAT TCTAC CTCTGTG GTCTCTGGAAACAAGCTCAAGCATTTT TA GCAGTGC ATC hTRB hTRB 205 ATGAACCCCAAACTCTTCTGTGTGACCCTTTGTCTCCTGGGAGCAGGC TCCCC GCACCAG V08-2 V08-2 TCTATTGATGCTGGGATCACCCAGATGCCAAGATATCACATTGTACAG AATCC CCAGACC AAGAAAGAGATGATCCTGGAATGTGCTCAGGTTAGGAACAGTGTTCTG TGGCA TATCTGT ATATCGACAGGACCCAAGACGGGGGCTGAAGCTTATCCACTATTCAGG TCCAC ACCACTG CAGTGGTCACAGCAGGACCAAAGTTGATGTCACAGAGGGGTACTGTGT CA TGGCAGC TTCTTGAAACAAGCTTGAGCATT ACATC hTRB hTRB 206 ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCA CTAAA GGGGGAC V09 V09 GGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACA CCTGA TCAGCTT GCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGAC GCTCT TGTATTT CTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTC CTGGA CTGTGCC CTCATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTT GCT AGCAGCG GAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAA TAG hTRB hTRB 207 ATGGGCACGAGGCTCTTCTTCTATGTGGCCCTTTGTCTGCTGTGGGCA CCCTC CTCCTCC V10-1 V10-1 GGACACAGGGATGCTGAAATCACCCAGAGCCCAAGACACAAGATCACA ACTCT CAGACAT GAGACAGGAAGGCAGGTGACCTTGGCGTGTCACCAGACTTGGAACCAC GGAGT CTGTATA AACAATATGTTCTGGTATCGACAAGACCTGGGACATGGGCTGAGGCTG CTGCT TTTCTGC ATCCATTACTCATATGGTGTTCAAGACACTAACAAAGGAGAAGTCTCA GC GCCAGCA GATGGCTACAGTGTCTCTAGATCAAACACAGAGGACCTCC GTGAGTC hTRB hTRB 208 ATGGGCACCAGGCTCTTCTTCTATGTGGCCCTTTGTCTGCTGTGGGCA CCCTC CCGCTCC V10-2 V10-2 GGACACAGGGATGCTGGAATCACCCAGAGCCCAAGATACAAGATCACA ACTCT CAGACAT GAGACAGGAAGGCAGGTGACCTTGATGTGTCACCAGACTTGGAGCCAC GGAGT CTGTGTA AGCTATATGTTCTGGTATCGACAAGACCTGGGACATGGGCTGAGGCTG CAGCT TTTCTGC ATCTATTACTCAGCAGCTGCTGATATTACAGATAAAGGAGAAGTCCCC AC GCCAGCA GATGGCTATGTTGTCTCCAGATCCAAGACAGAGAATTTCC GTGAGTC hTRB hTRB 209 ATGGGCACAAGGTTGTTCTTCTATGTGGCCCTTTGTCTCCTGTGGACA TCCTC CAGCTCC V10-3 V10-3 GGACACATGGATGCTGGAATCACCCAGAGCCCAAGACACAAGGTCACA ACTCT CAGACAT GAGACAGGAACACCAGTGACTCTGAGATGTCACCAGACTGAGAACCAC GGAGT CTGTGTA CGCTATATGTACTGGTATCGACAAGACCCGGGGCATGGGCTGAGGCTG CCGCT CTTCTGT ATCCATTACTCATATGGTGTTAAAGATACTGACAAAGGAGAAGTCTCA AC GCCATCA GATGGCTATAGTGTCTCTAGATCAAAGACAGAGGATTTCC GTGAGTC hTRB hTRB 210 ATGAGCACCAGGCTTCTCTGCTGGATGGCCCTCTGTCTCCTGGGGGCA CCACT GAGCTTG V11-1 V11-1 GAACTCTCAGAAGCTGAAGTTGCCCAGTCCCCCAGATATAAGATTACA CTCAA GGGACTC GAGAAAAGCCAGGCTGTGGCTTTTTGGTGTGATCCTATTTCTGGCCAT GATCC GGCCATG GCTACCCTTTACTGGTACCGGCAGATCCTGGGACAGGGCCCGGAGCTT AGCCT TATCTCT CTGGTTCAATTTCAGGATGAGAGTGTAGTAGATGATTCACAGTTGCCT GCA GTGCCAG AAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACT CAGCTTA GC hTRB hTRB 211 ATGGGCACCAGGCTCCTCTGCTGGGCGGCCCTCTGTCTCCTGGGAGCA CCACT AAGCTTG V11-2 V11-1 GAACTCACAGAAGCTGGAGTTGCCCAGTCTCCCAGATATAAGATTATA CTCAA AGGACTC GAGAAAAGGCAGAGTGTGGCTTTTTGGTGCAATCCTATATCTGGCCAT GATCC GGCCGTG GCTACCCTTTACTGGTACCAGCAGATCCTGGGACAGGGCCCAAAGCTT AGCCT TATCTCT CTGATTCAGTTTCAGAATAACGGTGTAGTGGATGATTCACAGTTGCCT GCA GTGCCAG AAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACT CAGCTTA GA hTRB hTRB 212 ATGGGTACCAGGCTCCTCTGCTGGGTGGCCTTCTGTCTCCTGGTGGAA CCACT GAGCTTG V11-3 V11-1 GAACTCATAGAAGCTGGAGTGGTTCAGTCTCCCAGATATAAGATTATA CTCAA GGGACTC GAGAAAAAACAGCCTGTGGCTTTTTGGTGCAATCCTATTTCTGGCCAC GATCC GGCCGTG AATACCCTTTACTGGTACCTGCAGAACTTGGGACAGGGCCCGGAGCTT AGCCT TATCTCT CTGATTCGATATGAGAATGAGGAAGCAGTAGACGATTCACAGTTGCCT GCA GTGCCAG AAGGATCGATTTTCTGCAGAGAGGCTCAAAGGAGTAGACT CAGCTTA GA hTRB hTRB 213 ATGGGCTCTTGGACCCTCTGTGTGTCCCTTTATATCCTGGTAGCGACA GAGGA GGGACTT V12-1 V12-1 CACACAGATGCTGGTGTTATCCAGTCACCCAGGCACAAAGTGACAGAG TCCAG GGGCCTA ATGGGACAATCAGTAACTCTGAGATGCGAACCAATTTCAGGCCACAAT CCCAT TATTTCT GATCTTCTCTGGTACAGACAGACCTTTGTGCAGGGACTGGAATTGCTG GGAAC GTGCCAG AATTACTTCTGCAGCTGGACCCTCGTAGATGACTCAGGAGTGTCCAAG CCA CAGCTTT GATTGATTCTCAGCACAGATGCCTGATGTATCATTCTCCACTCT GC hTRB hTRB 214 ATGGACTCCTGGACCCTCTGTGTGTCCCTTTGTATCCTGGTAGCGACA CTGAA AGGGGGA V12-2 V12-2 TGCACAGATGCTGGCATTATCCAGTCACCCAAGCATGAGGTGACAGAA GATCC CTCGGCC ATGGGACAAACAGTGACTCTGAGATGTGAGCCAATTTTTGGCCACAAT AGCCT GTGTATG TTCCTTTTCTGGTACAGAGATACCTTCGTGCAGGGACTGGAATTGCTG GCAGA TCTGTGC AGTTACTTCCGGAGCTGATCTATTATAGATAATGCAGGTATGCCCACA GC AAGTCGC GAGCGATTCTCAGCTGAGAGGCCTGATGGATCATTCTCTACT TTAGC hTRB hTRB 215 ATGGACTCCTGGACCTTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCG CAGCC CAGCTGT V12-3 V12-3 AAGCATACAGATGCTGGAGTTATCCAGTCACCCCGCCATGAGGTGACA CTCAG GTACTTC GAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGCCAC AACCC TGTGCCA AACTCCCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTG AGGGA GCAGTTT CTCATTTACTTTAACAACAACGTTCCGATAGATGATTCAGGGATGCCC CT AGC GAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTG AAGATC hTRB hTRB 216 ATGGGCTCCTGGACCCTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCA CAGCC CAGCTGT V12-4 V12-3 AAGCACACAGATGCTGGAGTTATCCAGTCACCCCGGCACGAGGTGACA CTCAG GTACTTC GAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGACAC AACCC TGTGCCA GACTACCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTG AGGGA GCAGTTT CTCATTTACTTTAACAACAACGTTCCGATAGATGATTCAGGGATGCCC CT AGC GAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTG AAGATC hTRB hTRB 217 ATGGCCACCAGGCTCCTCTGCTGTGTGGTTCTTTGTCTCCTGGGAGAA CAGCC CAGCTGT V12-5 V12-3 GAGCTTATAGATGCTAGAGTCACCCAGACACCAAGGCACAAGGTGACA CTCAG GTATTTT GAGATGGGACAAGAAGTAACAATGAGATGTCAGCCAATTTTAGGCCAC AACCC TGTGCTA AATACTGTTTTCTGGTACAGACAGACCATGATGCAAGGACTGGAGTTG AGGGA GTGGTTT CTGGCTTACTTCCGCAACCGGGCTCCTCTAGATGATTCGGGGATGCCG CT GGT AAGGATCGATTCTCAGCAGAGATGCCTGATGCAACTTTAGCCACTCTG AAGATC hTRB hTRB 218 ATGCTTAGTCCTGACCTGCCTGACTCTGCCTGGAACACCAGGCTCCTC GAGCT CAGCCCT V13 V13 TGCCATGTCATGCTTTGTCTCCTGGGAGCAGTTTCAGTGGCTGCTGGA CCTTG GTACTTC GTCATCCAGTCCCCAAGACATCTGATCAAAGAAAAGAGGGAAACAGCC GAGCT TGTGCCA ACTCTGAAATGCTATCCTATCCCTAGACACGACACTGTCTACTGGTAC GGGGG GCAGCTT CAGCAGGGTCCAGGTCAGGACCCCCAGTTCCTCATTTCGTTTTATGAA ACT AGG AAGATGCAGAGCGATAAAGGAAGCATCCCTGATCGATTCTCAGCTCAA CAGTTCAGTGACTATCATTCTGAACTGAACAT hTRB hTRB 219 ATGGTTTCCAGGCTTCTCAGTTTAGTGTCCCTTTGTCTCCTGGGAGCA GGTGC GATTCTG V14 V14 AAGCACATAGAAGCTGGAGTTACTCAGTTCCCCAGCCACAGCGTAATA AGCCT GAGTTTA GAGAAGGGCCAGACTGTGACTCTGAGATGTGACCCAATTTCTGGACAT GCAGA TTTCTGT GATAATCTTTATTGGTATCGACGTGTTATGGGAAAAGAAATAAAATTT ACTGG GCCAGCA CTGTTACATTTTGTGAAAGAGTCTAAACAGGATGAGTCCGGTATGCCC AG GCCAAGA AACAATCGATTCTTAGCTGAAAGGACTGGAGGGACGTATTCTACTCTG AA hTRB hTRB 220 ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACA GACAT GGGACAC V15 V15 GGTCATGGGGATGCCATGGTCATCCAGAACCCAAGATACCAGGTTACC CCGCT AGCCATG CAGTTTGGAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCAT CACCA TACCTGT AACGTCATGTACTGGTACCAGCAGAAGTCAAGTCAGGCCCCAAAGCTG GGCCT GTGCCAC CTGTTCCACTACTATGACAAAGATTTTAACAATGAAGCAGACACCCCT GG CAGCAGA GATAACTTCCAATCCAGGAGGCCGAACACTTCTTTCTGCTTTCTT GA hTRB hTRB 221 ATGAGCCCAATATTCACCTGCATCACAATCCTTTGTCTGCTGGCTGCA TGAGA GAGGATT V16 V16 GGTTCTCCTGGTGAAGAAGTCGCCCAGACTCCAAAACATCTTGTCAGA TCCAG CAGCAGT GGGGAAGGACAGAAAGCAAAATTATATTGTGCCCCAATAAAAGGACAC GCTAC GTATTTT AGTTATGTTTTTTGGTACCAACAGGTCCTGAAAAACGAGTTCAAGTTC GAAGC TGTGCCA TTGATTTCCTTCCAGAATGAAAATGTCTTTGATGAAACAGGTATGCCC TT GCAGCCA AAGGAAAGATTTTCAGCTAAGTGCCTCCCAAATTCACCCTGTAGCCT ATC hTRB hTRB 222 ATGGATATCTGGCTCCTCTGCTGGGTGACCCTGTGTCTCTTGGCGGCA GAAGA AGGGACT V17 V17 GGACACTCGGAGCCTGGAGTCAGCCAGACCCCCAGACACAAGGTCACC TCCAT CAGCCGT AACATGGGACAGGAGGTGATTCTGAGGTGCGATCCATCTTCTGGTCAC CCCGC GTATCTC ATGTTTGTTCACTGGTACCGACAGAATCTGAGGCAAGAAATGAAGTTG AGAGC TACAGTA CTGATTTCCTTCCAGTACCAAAACATTGCAGTTGATTCAGGGATGCCC CG GCGGTGG AAGGAACGATTCACAGCTGAAAGACCTAACGGAACGTCTTCCACGCT hTRB hTRB 223 ATGGACACCAGAGTACTCTGCTGTGCGGTCATCTGTCTTCTGGGGGCA GGATC AGATTCG V18 V18 GGTCTCTCAAATGCCGGCGTCATGCAGAACCCAAGACACCTGGTCAGG CAGCA GCAGCTT AGGAGGGGACAGGAGGCAAGACTGAGATGCAGCCCAATGAAAGGACAC GGTAG ATTTCTG AGTCATGTTTACTGGTATCGGCAGCTCCCAGAGGAAGGTCTGAAATTC TGCGA TGCCAGC ATGGTTTATCTCCAGAAAGAAAATATCATAGATGAGTCAGGAATGCCA GG TCACCAC AAGGAACGATTTTCTGCTGAATTTCCCAAAGAGGGCCCCAGCATCCTG C A hTRB hTRB 224 ATGAGCAACCAGGTGCTCTGCTGTGTGGTCCTTTGTTTCCTGGGAGCA CACTG AACCCGA V19 V19 AACACCGTGGATGGTGGAATCACTCAGTCCCCAAAGTACCTGTTCAGA TGACA CAGCTTT AAGGAAGGACAGAATGTGACCCTGAGTTGTGAACAGAATTTGAACCAC TCGGC CTATCTC GATGCCATGTACTGGTACCGACAGGACCCAGGGCAAGGGCTGAGATTG CCAAA TGTGCCA ATCTACTACTCACAGATAGTAAATGACTTTCAGAAAGGAGATATAGCT AG GTAGTAT GAAGGGTACAGCGTCTCTCGGGAGAAGAAGGAATCCTTTCCTCT AGA hTRB hTRB 225 ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGGTATAAGCCTCCTTCTA CTGAC CTGAAGA V20 V20 CCTGGGAGCTTGGCAGGCTCCGGGCTTGGTGCTGTCGTCTCTCAACAT AGTGA CAGCAGC CCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGC CCAGT TTCTACA CGTTCCCTGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTC GCCCA TCTGCAG CCGAAACAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAG TC TGCTAGA GCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCAT GA GCAAGCCTGACCTTGTCCACT hTRB hTRB 226 ATGTGCCTCAGACTTCTCTGCTGTGTGGCCATTTCTTTCTGGGGAGCC GAGAT GGGACAC V21 V21 AGGCTCCACGGACACCAAGGTCACCCAGAGACCTAGACTTCTGGTCAA CCAGT AGCACTG AGCAAGTGAACAGAAAGCAAAGATGGATTGTGTTCCTATAAAAGCACA CCACG TATTTCT TAGTTATGTTTACTGGTATCGTAAGAAGCTGGAAGAAGAGCTCAAGTT GAGTC GTGCCAG TTTGGTTTACTTTCAGAATGAAGAACTTATTCAGAAAGCAGAAATAAT AG CAGCAAA CAATGAGCGATTTTTAGCCCAATGCTCCAAAAACTCATCCTGTACCTT GC G hTRB hTRB 227 ATGGGGAGCTGGGTCCTCTGCTATGTGACCCTGTGTCTCCTGGGAGCA GTGAA AACAGCT V22 V22 GGACCCTTGGATGCTGACATCTATCAGATGCCATTCCAGCTCACTGGG GTTGG TTGTACT GCTGGATGGGATGTGACTCTGGAGTGGAAACGGAATTTGAGACACAAT CCCAC TCTGTCC GACATGTACTGCTACTGGTACTGGCAGGACCCAAAGCAAAATCTGAGA ACCAG TGGGAGC CTGATCTATTACTCAAGGGTTGAAAAGGATATTCAGAGAGGAGATCTA CCA GCAC ACTGAAGGCTACGTGTCTGCCAAGAGGAGAAGGGGCTATTTCTTCTCA GG hTRB hTRB 228 ATGGGCACCAGGCTCCTCGGCTGTGCAGCCCTGTGTCTCCTGGCAGCA CCTGG CCGGGAG V23 V23 GACTCTTTTCATGCCAAAGTCACACAGACTCCAGGACATTTGGTCAAA CAATC ACACGGC GGAAAAGGACAGAAAACAAAGATGGATTGTACCCCCGAAAAAGGACAT CTGTC ACTGTAT ACTTTTGTTTATTGGTATCAACAGAATCAGAATAAAGAGTTTATGCTT CTCAG CTCTGCG TTGATTTCCTTTCAGAATGAACAAGTTCTTCAAGAAACGGAGATGCAC AA CCAGCAG AAGAAGCGATTCTCATCTCAATGCCCCAAGAACGCACCCTGCAG TCAATC hTRB hTRB 229 ATGGCCTCCCTGCTCTTCTTCTGTGGGGCCTTTTATCTCCTGGGAACA GAGTC CAGCTCT V24 V24 GGGTCCATGGATGCTGATGTTACCCAGACCCCAAGGAATAGGATCACA TGCCA TTACTTC AAGACAGGAAAGAGGATTATGCTGGAATGTTCTCAGACTAAGGGTCAT TCCCC TGTGCCA GATAGAATGTACTGGTATCGACAAGACCCAGGACTGGGCCTACGGTTG AACCA CCAGTGA ATCTATTACTCCTTTGATGTCAAAGATATAAACAAAGGAGAGATCTCT GA TTTG GATGGATACAGTGTCTCTCGACAGGCACAGGCTAAATTCTCCCTGTCC CTA hTRB hTRB 230 ATGACTATCAGGCTCCTCTGCTACATGGGCTTTTATTTTCTGGGGGCA GGAGT TACCTCT V25 V25 GGCCTCATGGAAGCTGACATCTACCAGACCCCAAGATACCTTGTTATA CTGCC CAGTACC GGGACAGGAAAGAAGATCACTCTGGAATGTTCTCAAACCATGGGCCAT AGGCC TCTGTGC GACAAAATGTACTGGTATCAACAAGATCCAGGAATGGAACTACACCTC CTCAC CAGCAGT ATCCACTATTCCTATGGAGTTAATTCCACAGAGAAGGGAGATCTTTCC A GAATA TCTGAGTCAACAGTCTCCAGAATAAGGACGGAGCATTTTCCCCTGACC CT hTRB hTRB 231 ATGAGCAACAGGCTTCTCTGCTGTGTGATCATTTGTCTCCTAAGAGCA GAAGT ACATCTG V26 V26 GGCCTCAAGGATGCTGTAGTTACACAATTCCCAAGACACAGAATCATT CTGCC TGTATCT GGGACAGGAAAGGAATTCATTCTACAGTGTTCCCAGAATATGAATCAT AGCAC CTATGCC GTTACAATGTACTGGTATCGACAGGACCCAGGACTTGGACTGAAGCTG CAACC AGCAGTT GTCTATTATTCACCTGGCACTGGGAGCACTGAAAAAGGAGATATCTCT AG CATC GAGGGGTATCATGTTTCTTGAAATACTATAGCATCTTTTCCCCTGACC CT hTRB hTRB 232 ATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCA GGAGT ACCTCTC V27 V27 GGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACA CGCCC TGTACTT GTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCAT AGCCC CTGTGCC GAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAG CAACC AGCAGTT ATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCT AG TATC GAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATC CT hTRB hTRB 233 ATGGGAATCAGGCTCCTGTGTCGTGTGGCCTTTTGTTTCCTGGCTGTA GGAGT ACATCTA V28 V28 GGCCTCGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAA CCGCC TGTACCT AGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCAT AGCAC CTGTGCC GAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTG CAACC AGCAGTT ATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCT AG TATG GAGGGGTACAGTGTCTCTAGAGAGAAGAAGGAGCGCTTCTCCCTGATT CT hTRB hTRB 234 ATGCTGAGTCTTCTGCTCCTTCTCCTGGGACTAGGCTCTGTGTTCAGT GTGAG CAGCAGC V29 V29 GCTGTCATCTCTCAAAAGCCAAGCAGGGATATCTGTCAACGTGGAACC CAACA ATATATC TCCCTGACGATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTC TGAGC TCTGCAG TGGTACCGTCAGCAACCTGGACAGAGCCTGACACTGATCGCAACTGCA CCTGA CGTTGAA AATCAGGGCTCTGAGGCCACATATGAGAGTGGATTTGTCATTGACAAG AGA GA TTTCCCATCAGCCGCCCAAACCTAACATTCTCAACTCTGACT hTRB hTRB 235 ATGCTCTGCTCTCTCCTTGCCCTTCTCCTGGGCACTTTCTTTGGGGTC GAGTT GTGACTC V30 V30 AGATCTCAGACTATTCATCAATGGCCAGCGACCCTGGTGCAGCCTGTG CTAAG TGGCTTC GGCAGCCCGCTCTCTCTGGAGTGCACTGTGGAGGGAACATCAAACCCC AAGCT TATCTCT AACCTATACTGGTACCGACAGGCTGCAGGCAGGGGCCTCCAGCTGCTC CCTTC GTGCCTG TTCTACTCCGTTGGTATTGGCCAGATCAGCTCTGAGGTGCCCCAGAAT TCA GAGTGT CTCTCAGCCTCCAGACCCCAGGACCGGCAGTTCATCCT 

1.-30. (canceled)
 31. A method for sequencing immune cell receptor genes, comprising providing RNA from immune cells; (a) (1) transcribing the RNA into complementary RNA (cRNA), using one or more primers wherein the cRNA is produced by transcription; (2) reverse transcribing the cRNA into single strand DNA (ssDNA), using one or more primers that comprise a 5′ adapter sequence, wherein each 5′ end of the ssDNA produced by reverse transcription contains the 5′ adapter sequence; or (b) (1) transcribing the RNA into complementary RNA (cRNA), using one or more primers that comprise a 3′ adapter sequence, wherein each 3′ end of the cRNA produced by transcription contains the 3′ adapter sequence; (2) reverse transcribing the cRNA into single strand DNA (ssDNA), using one or more primers that comprise a 5′ adapter sequence, wherein each 5′ end of the ssDNA produced by reverse transcription contains the 5′ adapter sequence and wherein each 3′ end of the ssDNA produced by reverse transcription contains the 3′ adapter sequence; amplifying the ssDNA to produce a first amplification product using a first primer pair comprising a first primer that hybridizes to the 5′ adapter sequence and a second primer that hybridizes to a constant region of immune cell receptor gene or to the 3′ adapter sequence; amplifying the first amplification product to produce a second amplification product using a second primer pair, in which i. a first primer of the second primer pair binds to the adapter sequence at the 5′ end of the first amplification product, ii. the second primer of the second primer pair binds to the constant region of immune cell receptor gene in the first amplification product, and iii. the first and second primers comprise adapter sequences for sequencing; and sequencing the second amplification product.
 32. The method according to claim 31, wherein the reverse transcription step results in PCR products ranging from 150-600 bp.
 33. The method according to claim 31, wherein the immune cell receptor genes are T-cell receptor (TCR) genes or B-cell receptor (BCR) genes.
 34. The method according to claim 31, wherein the one or more primers used for reverse transcription hybridize to TCR α chain V segments, optionally wherein the one or more primers used for reverse transcription comprise one or more of SEQ ID NOs: 1-50; or wherein the one or more primers used for reverse transcription hybridize to TCR β chain V segments, optionally wherein the one or more primers used for reverse transcription comprise one or more of SEQ ID NOs: 51-100; or wherein the one or more primers used for reverse transcription hybridize to TCR γ chain V segments; or wherein the one or more primers used for reverse transcription hybridize to TCR β chain V segments; or wherein the one or more primers used for reverse transcription hybridize to BCR heavy chain V segments; or wherein the one or more primers used for reverse transcription hybridize to BCR light chain V segments.
 35. The method according to claim 31, wherein the one or more primers used for reverse transcription contain a nucleotide barcode sequence; optionally wherein the nucleotide barcode comprises 6 to 20 nucleotides.
 36. The method according to claim 35, wherein the nucleotide barcode consists of 9 nucleotides; optionally wherein the nucleotide barcode consists of the sequence NNNNTNNNN, NNNNANNNN or HHHHHNNNN.
 37. The method according to claim 31, wherein the 5′ adapter sequence of the one or more primers used for the reverse transcription comprises a T7 adapter.
 38. The method according to claim 31, wherein the immune cells are T-cells and wherein the second primer of the first pair of primers hybridizes to the constant region of a TCR gene.
 39. The method according to claim 31, wherein the immune cells are B-cells and wherein the second primer of the first pair of primers hybridizes to the constant region of a BCR gene.
 40. The method according to claim 31, wherein the sequencing is next generation sequencing.
 41. The method according to claim 31, wherein the RNA from the immune cells is obtained by mixing immune cells with carrier cells before RNA extraction.
 42. The method according to claim 31, wherein the immune cells are tumor-infiltrating lymphocytes.
 43. The method according to claim 31, wherein the immune cells are CD4 or CD8 positive T-cells.
 44. The method according to claim 31, wherein the immune cells are purified from peripheral blood mononuclear cells (PBMC) before RNA extraction.
 45. The method according to claim 31, wherein the immune cells are part of a mixture of peripheral blood mononuclear cells (PBMC).
 46. The method according to claim 31, wherein the immune cells are derived from a mammal; optionally wherein the mammal is a human or a mouse.
 47. A kit for sequencing of T-cell receptors (TCRs), comprising: (a) at least one primer which comprises a TCR α chain V segment and a barcode sequence; or (b) at least one primer which comprises a TCR β chain V segment and a barcode sequence.
 48. The kit according to claim 47, (a) wherein the at least one primer which comprises a TCR α chain V segment and a barcode sequence comprises any one of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310; or (b) wherein the at least one primer which comprises a TCR β chain V segment and a barcode sequence comprises any one of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360.
 49. The kit according to claim 48, wherein the kit comprises at least one primer which comprises a sequence of any one of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310.
 50. The kit according to claim 48, wherein the kit comprises at least one primer which comprises a sequence of any one of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360. 